Peptides and combination of peptides and scaffolds for use in immunotherapy against Renal Cell Carcinoma (RCC) and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/978,700, filed May 14, 2018, which is a continuation of U.S.application Ser. No. 15/799,495, filed Oct. 31, 2017, now U.S. Pat. No.10,105,428, issued Oct. 23, 2018, which is a continuation of U.S.application Ser. No. 15/082,920, filed Mar. 28, 2016, now U.S. Pat. No.9,931,388, issued Apr. 3, 2018, which claims the benefit of U.S.Provisional Application Ser. No. 62/140,767, filed Mar. 31, 2015, andGreat Britain Application No. 1505585.8, filed Mar. 31, 2015, thecontent of each these applications is herein incorporated by referencein their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.TXT)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “2912919-043006_SEQ_LIST.txt,” created on Dec. 5,2018, and 23,850 bytes in size) is submitted concurrently with theinstant application, and the entire contents of the Sequence Listing areincorporated herein by reference.

FIELD

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

Kidney cancer is more common in men than women and is the ninth mostcommon cancer in men (214 000 cases) and the 14th most common in women(124 000 cases) worldwide in 2012. 70% of the new cases occurred incountries with high and very high levels of human development, with 34%of the estimated new cases in Europe and 19% in North America. Therewere an estimated 143 000 deaths from kidney cancer in 2012 (91 000 inmen, 52 000 in women); kidney cancer is the 16th most common cause ofdeath from cancer worldwide.

The highest incidence rates are found in the Czech Republic. Elevatedrates are also found in northern and Eastern Europe, North America, andAustralia. Low rates are estimated in much of Africa and East Asia. Thecase fatality rate is lower in highly developed countries (overallmortality-to-incidence ratio, 0.4) than in countries with low or mediumlevels of human development (0.5). Only 3.1% of the cases were diagnosedin Africa, but 5.7% of the deaths occurred in this region. Incidence andmortality rates have been increasing in many countries, across differentlevels of human development (World Cancer Report, 2014).

Most renal cancers are renal cell carcinomas (RCC), a heterogeneousclass of tumors arising from different cell types within the renalparenchyma. Most are clear cell renal carcinomas (about 70% of renalcancer cases), followed by papillary (10-15%), chromophobe (about 5%),and collecting duct (<1° A) renal cell carcinomas. Each of these renalcell tumor subtypes has distinct genetic characteristics (Moch, 2013;World Health Organization Classification of Tumours, 2004).

Renal cell carcinoma (RCC) is characterized by a lack of early warningsigns, diverse clinical manifestations, and resistance to radiation andchemotherapy. A total of 25-30% patients with RCC initially present withovert metastases (Hofmann et al., 2005). About one third of patientswith RCC will develop metastatic disease over time. Thus, nearly 50-60%of all patients with RCC will eventually present with metastatic disease(Bleumer et al., 2003; Hofmann et al., 2005). Among those withmetastatic disease, approximately 75% have lung metastases, 36% lymphnode and/or soft tissue involvement, 20% bone involvement, and 18% liverinvolvement (Sachdeva et al., 2010).

RCC patients with metastatic disease receiving cytokine-based first-linesystemic therapy can be categorized into risk groups predictive forsurvival based on 5 prognostic factors (Motzer et al., 2004).Pre-treatment features associated with a shorter survival were lowKarnofsky Performance Status (<80%), high serum lactate dehydrogenase(>1.5 ULN), low hemoglobin (<LLN), high corrected serum calcium (>10mg/dL), and time from diagnosis to treatment<1 year. Based on these riskfactors, patients were categorized into three risk groups. The mediantime to death in the 18% of patients with zero risk factors(favorable-risk) was 30 months. 62% of the patients had one or two riskfactors (intermediate risk), and the median survival time in this groupwas 14 months. Patients with 3 or more risk factors (poor risk) whocomprised 20% of the patients, had a median survival time of 5 months.The application of this MSKCC risk group categorization has been widelyapplied in clinical trials for advanced RCC. Risk categorization can beused for planning and interpreting the results of clinical trials anddirecting therapy.

Risk factors for RCC are cigarette smoking and obesity. Differentmeta-analyses confirmed that ever-smoking increases the risk of renalcancer compared with never-smoking (Cho et al., 2011; Hunt et al.,2005). There is also a dose-dependent increase in risk related to thenumber of cigarettes smoked per day. Risk decreases in the 5-year periodafter smoking cessation. Overweight, especially obesity, is a riskfactor for renal cancer in both women and men (Ljungberg et al., 2011).The proportion of all cases of renal cancer attributable to overweightand obesity has been estimated to be about 40% in the USA and up to 40%in European countries (Renehan et al., 2008; Renehan et al., 2010). Themechanisms by which obesity influences renal carcinogenesis are unclear.Sex steroid hormones may affect renal cell proliferation by directendocrine receptor-mediated effects. Obesity with the combined endocrinedisorders, such as decreased levels of sex hormonebinding globulin andprogesterone, insulin resistance, and increased levels of growth factorssuch as insulin-like growth factor 1 (IGF-1), may contribute to renalcarcinogenesis. Recently, a case-control study has reported a strongerassociation of clear cell carcinoma with obesity (World Cancer Report,2014).

Initial treatment is most commonly either partial or complete removal ofthe affected kidney(s) and remains the mainstay of curative treatment(Rini et al., 2008). For first-line treatment of patients with poorprognostic score a guidance elaborated by several cancer organizationsand societies recommend the receptor tyrosine kinase inhibitors (TKIs)sunitinib (Sutent®) and pazopanib (Votrient®), the monoclonal antibodybevacizumab (Avastin®) combined with interferon-α (IFN-α) and the mTORinhibitor temsirolimus (Torisel®). Based on guidelines elaborated by theUS NCCN as well as the European EAU and ESMO, the TKIs sorafenib,pazopanib or recently axitinib are recommended as second-line therapy inRCC patients who have failed prior therapy with cytokines (IFN-α, IL-2).The NCCN guidelines advise also sunitinib in this setting (high-levelevidence according to NCCN Category I).

Everolimus and axitinib are recommended as second-line therapy of thosepatients who have not benefited from a VEGF-targeted therapy with TKIsaccording to the established guidelines.

The known immunogenity of RCC has represented the basis supporting theuse of immunotherapy and cancer vaccines in advanced RCC.

The interesting correlation between lymphocytes PD-1 expression and RCCadvanced stage, grade and prognosis, as well as the selective PD-L1expression by RCC tumor cells and its potential association with worseclinical outcomes, have led to the development of new anti PD-1/PD-L1agents, alone or in combination with anti-angiogenic drugs or otherimmunotherapeutic approaches, for the treatment of RCC (Massari et al.,2015).

In advanced RCC, a phase III cancer vaccine trial called TRIST studyevaluates whether TroVax (a vaccine using a tumor-associated antigen,5T4, with a pox virus vector), added to first-line standard of caretherapy, prolongs survival of patients with locally advanced or mRCC.Median survival had not been reached in either group with 399 patients(54%) remaining on study however analysis of the data confirms priorclinical results, demonstrating that TroVax is both immunologicallyactive and that there is a correlation between the strength of the5T4-specific antibody response and improved survival. Further there areseveral studies searching for Peptide vaccines usind Epitopes beingoverexpressed in RCC.

Variuos approaches of tumor vaccines have been under investigation.Studies using whole-tumor approaches, including tumor cell lysates,fusions of dendritic cells with tumor cells, or whole-tumor RNA weredone in RCC patients, and remissions of tumor lesions were reported insome of thesetrials (Avigan et al., 2004; Holtl et al., 2002; Marten etal., 2002; Su et al., 2003; Wittig et al., 2001).

In the last years, several human TAAs expressed in RCCs and recognizedby antigen-specific CTLs have been defined and characterized usingexpression cloning, reverse immunology approach, or by applying DNAmicroarray technology (Dannenmann et al., 2013; Michael and Pandha,2003; Minami et al., 2014; Renkvist et al., 2001; Wierecky et al.,2006).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and RCC in particular. There is also aneed to identify factors representing biomarkers for cancer in generaland RCC in particular, leading to better diagnosis of cancer, assessmentof prognosis, and prediction of treatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor- (-associated) exon in case of proteins with tumor-specific(-associated) isoforms.e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated (longer) peptides of the invention can act as MHC class IIactive epitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014a).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+ peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+ peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-I-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 114 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 114, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 114 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 114,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 1 and Table2 bind to HLA-A*02. The peptides in Table 2 have been disclosed beforein large listings as results of high-throughput screenings with higherror rates or calculated using algorithms, but have not been associatedwith cancer at all before. The peptides in Table 3 are additionalpeptides that may be useful in combination with the other peptides ofthe invention. The peptides in Tables 4A and B are furthermore useful inthe diagnosis and/or treatment of various other malignancies thatinvolve an over-expression or over-presentation of the respectiveunderlying polypeptide.

TABLE 1 Peptides according to the present invention SEQ ID Official GeneNo Sequence Gene ID(s) Symbol(s) 1 ALIVSLPYL     10786 SLC17A3 2ILWREVVTL      3299 HSF4 3 RLLGEVQAL      3299 HSF4 4 FLSQDIITV     5972 REN 5 YLYPNLTRL      6540 SLC6A13 6 VLFELSKTV     23250 ATP11A7 FLLSLIDRL    112399 EGLN3 8 GLASFKSFL      8490 RGS5 9 ILLQKPDSV     8490 RGS5 10 KLLQNNYGL      8490 RGS5 11 FIQTEAPKEV      8490 RGS512 ALDPSGNQLI     54437 SEMA5B 13 KIMAQILTV    120892 LRRK2 14 ALLTETIFL   120892 LRRK2 15 ILIKHLVKV    143872 ARHGAP42 16 FMPEELPQL     55258THNSL2 17 ILAQQVHAL    113220 KIF12 18 YVLDLAAKV        47 ACLY 19LLDPGSLQL    646658 SYNDIG1L 20 AVANTTFTV     80270 HSD3B7 21RLIQGDQILSV     10207 INADL 22 FLSPPLPSV       593, BCKDHA, TMEM91   641649 23 YIQEVVQYI     23236 PLCB1 24 FTLGTTVFL      4717 NDUFC1 25LLVPAHLVAA     11082 ESM1 26 SLMEILYTL     91949 COG7 27 SLSDLLVSL    23596 OPN3 28 FIADLVVGL      2023, ENO1, ENO2, ENO3      2026,     2027 29 ILLDLEQAL      9820 CUL7 30 QLFYTKIFL      5351 PLOD1 31VLFGLDPAVIKV    259217 HSPA12A 32 FLAGGIRGSGA    113730 KLHDC7B 33FIADVVEKI      5654, HTRA1, HTRA3     94031 34 ELNNQNFYL     11113 CIT35 VLHSLQTQL     51129 ANGPTL4 36 SLFGKKYIL      2274 FHL2 37 VLAPVILML     8714 ABCC3 38 VLLDTILQL     11077 HSF2BP 39 YLLNLNHLGL     23471TRAM1 40 YIQEHLLQI     10625 IVNS1ABP 41 GLLKTLQKL     25932 CLIC4 42VILDTGTIQL      9027 NAT8 43 YLKDELDEL     23255 SOGA2 44 ALFSFVTAL    80727 TTYH3 45 ALLGIPLTL      3777, KCNK3, KCNK15     60598 46GLSEVLVQI     57553 MICAL3 47 TLAEVRAVQEI     56950 SMYD2 48 VVASNIMEV     5209 PFKFB3 49 VLIVEVPGV       111 ADCY5 50 SLSDHIVLL      3675ITGA3 51 NLWPMILTL      3675 ITGA3 52 SILDAVQRV    137902 PXDNL 53FLLEIRQTL     23161 SNX13 54 ALVAKGLVQA     10327 AKR1A1 55 YLALILPVL     9122 SLC16A4 56 ILMDFSNSM      3691 ITGB4 57 SLQKEILYL     55102ATG2B 58 FLVDFEQSHL      1573 CYP2J2 59 SLKNNVVSV      7045 TGFBI 60ILWKDIEYV    143425 SYT9 61 SLMGILLRI     22900 CARD8 62 VLAGPAFLVQL    55244 SLC47A1 63 GLIEDHFDVTV     51752 ERAP1 64 LLAASVALA      4885NPTX2 65 IIYGGSVTGA      7167, TPI1, TPI1P1    729708 66 TLLKTIIKV    57545 CC2D2A 67 LLDVLAPLV     80781 COL18A1 68 YVLTQPPSV     28796,IGLV3-21, IGLV2-14,     28815, IGLJ3, IGLC1, IGLC2     28831,      3537,     3538 69 ILADLLPSL     25979 DHRS7B 70 SLTALRLLL      9920 KBTBD1171 ALDGHLYAV      9920 KBTBD11 72 YSLEKVFGI     10916 MAGED2 73GLDGIPFTV      7205 TRIP6 74 GLFHKQVTV     23037 PDZD2 75 FLIKSINLV   143879 KBTBD3 76 VLADDHLIEV 100034743, PDZK1P2, PDZK1,      5174,PDZK1P1    728939 77 SLIKHKIML       523 ATP6V1A 78 ALLDTVVQA      8911,CACNA1I, CACNA1H      8912 79 ALADIVWRA     84182 FAM188B 80 KLASMLETL   112464 PRKCDBP 81 SLLPALPKL      4036 LRP2 82 SLLQATDFMSL      7070THY1 83 IQWSIVPEV     23151 GRAMD4 84 YLMDEGAHL      7358 UGDH 85FVMSEIRTV    114991 ZNF618 86 GLLQGKLALL      4835 NQO2 87 LADGVQKV     8542 APOL1 88 TLAELHISL     84166 NLRC5 89 SLLLAVTEV      3714 JAG290 FTLEKNFVI      1292 COL6A2 91 MLLSSLVSL     79001 VKORC1 92 FLFRDILEL    29102 DROSHA

TABLE 2 Additional peptides according to the presentinvention with no prior known cancer association SEQ ID No SequenceGene ID(s) Official Gene Symbol(s) 93 GVMAGDIYSV    123 PLIN2 94ILHHKVYDL   1528 CYB5A 95 KLTDVGIATL 115701 ALPK2 96 TLAETLVNL 283372,ANKRD52 283373 97 TLISELVQA   9820 CUL7 98 KIPPVSPSI  57561 ARRDC3 99GLAPHLEQI  79711 IPO4 100 KLNVAPLAV 653784, MZT2A, MZT2B  80097 101HIYDKAFITV   2321 FLT1 102 LLFDVHTTL  65250 C5orf42 103 KLQDGLLHI   7076TIMP1 104 ALFEGVVRQI   6236 RRAD 105 ALADLDELLIRA   3339 HSPG2 106VLMDLKALL  51428 DDX41 107 VLMDLKALLL  51428 DDX41 108 VLISVLQAI  26999CYFIP2 109 YLWSRVEKL 120892 LRRK2 110 LLDLHSYLL  10299 MARCH6 111TLLETEMLL  80817 CEP44 112 LLFDHLEPIEL  25780 RASGRP3 113 SLFDWNVKL134111 UBE2QL1 114 ALAVNISAA    908 CCT6A

TABLE 3 Peptides useful for e.g. personalized cancer therapies SEQ ID NoSequence Gene ID(s) Official Gene Symbol(s) 115 LLDPKTIFL     26762HAVCR1 116 GLVDIMVHL      8701 DNAH11 117 VLFGELPAL      8701 DNAH11 118FLNAIETAL      8701 DNAH11 119 RLHDENILL     23322 RPGRIP1L 120GLAGDNIYL      6582 SLC22A2 121 ALLRTVVSV      2590 GALNT2 122 SLDPSSPQV     9514 GAL3ST1 123 YVDPVITSI      4233 MET 124 ILSPLSVAL      5345SERPINF2 125 KLDPTKTTL     10397 NDRG1 126 KIQEILTQV     10643 IGF2BP3127 VLAPLFVYL      2535, FZD2, FZD1, FZD7      8321,      8324 128YLEEDVYQL     23255 SOGA2 129 VLAPRVLRA      5954 RCN1 130 ALPTVLVGV     5351 PLOD1 131 VMAGDIYSV       123 PLIN2 132 SVASTITGV       123PLIN2 133 QLIDYERQL     11072 DUSP14 134 VADKIHSV     11072 DUSP14 135VVDEGPTGV      9123 SLC16A3 136 YQDPHSTAV      1956 EGFR 137 TLVAIVVGV    60681 FKBP10 138 SLDTLMTYV     22829 NLGN4Y 139 ILNVDGLIGV        47ACLY 140 SLANNVTSV    131566 DCBLD2 141 LLVDDSFLHTV    253982 ASPHD1 142SVDVSPPKV    113146 AHNAK2 143 ALFVRLLALA      7045 TGFBI 144 RLLDVLAPLV    80781 COL18A1 145 SLHFLILYV       487, ATP2A1, ATP2A2       488 146KLIDLSQVMYL    346389 MACC1 147 ALADKELLPSV     84883 AIFM2 148KLLTEVHAA       101 ADAM8 149 SILTIEDGIFEV 100287551, HSPA8P8, HSPA2,     3306, HSPA8      3312 150 TLMPNINKL      5169 ENPP3 151 YMYEGPAPRI     5169 ENPP3

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, lung cancer, brain cancer,stomach cancer, colon or rectal cancer, liver cancer, pancreatic cancer,prostate cancer, leukemia, breast cancer, melanoma, ovarian cancer, andesophageal cancer.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 114. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 63 (see Table 1), and their uses in theimmunotherapy of RCC, lung cancer, brain cancer, stomach cancer, colonor rectal cancer, liver cancer, pancreatic cancer, prostate cancer,leukemias, breast cancer, melanoma, ovarian cancer, and esophagealcancer, and preferably RCC.

As shown in the following Table 4A, many of the peptides according tothe present invention are also found on other tumor types and can, thus,also be used in the immunotherapy of other indications. Also refer toFIG. 1E and Example 1.

TABLE 4APeptides according to the present invention and their specific uses in otherproliferative diseases, especially in other cancerous diseases. The table shows forselected peptides on which additional tumor types they were found and either over-presented on more than 5% of the measured tumor samples, or presented on more than5% of the measured tumor samples with a ratio of geometric means tumor vs normaltissues being larger than 3. Over-presentation is defined as higher presentation onthe tumor sample as compared to the normal sample with highest presentation.SEQ ID No Sequence Other relevant organs/diseases 1 ALIVSLPYL Liver 2ILWREVVTL Ovary 5 YLYPNLTRL Liver 8 GLASFKSFL Pancreas 12 ALDPSGNQLIOvary 14 ALLTETIFL Leukocytes 15 ILIKHLVKV Liver 16 FMPEELPQLPancreas, Breast, Ovary 21 RLIQGDQILSV Lung, Colon, Rectum, Liver, Ovary23 YIQEVVQYI Liver 24 FTLGTTVFL Liver, Prostate, Leukocytes, Esophagus26 SLMEILYTL Colon, Rectum, Pancreas, Prostate, Ovary 27 SLSDLLVSLLung, Liver, Pancreas, Leukocytes 28 FIADLVVGL Melanoma 29 ILLDLEQALLung, Pancreas, Prostate, Breast, Ovary 30 QLFYTKIFLLung, Colon, Rectum, Ovary 31 VLFGLDPAVIKV Brain, Ovary 32 FLAGGIRGSGAStomach 33 FIADVVEKI Lung, Leukocytes, Ovary 34 ELNNQNFYLMelanoma, Esophagus 36 SLFGKKYIL Colon, Rectum 37 VLAPVILMLLung, Colon, Rectum, Pancreas, Ovary 38 VLLDTILQLBrain, Liver, Pancreas, Melanoma, Ovary 39 YLLNLNHLGLLung, Liver, Leukocytes 44 ALFSFVTAL Lung, Melanoma, Ovary 45 ALLGIPLTLProstate, Ovary, Esophagus 46 GLSEVLVQI Colon, Rectum 47 TLAEVRAVQEILiver, Melanoma 49 VLIVEVPGV Ovary 50 SLSDHIVLL Lung 51 NLWPMILTLLung, Pancreas, Esophagus 52 SILDAVQRV Lung, Brain, Pancreas, Ovary 54ALVAKGLVQA Melanoma, Ovary 55 YLALILPVL Melanoma 56 ILMDFSNSM Pancreas59 SLKNNVVSV Pancreas, Ovary, Esophagus 61 SLMGILLRI Leukocytes 63GLIEDHFDVTV Lung, Colon, Rectum, Prostate, Leukocytes, Melanoma, Ovary65 IIYGGSVTGA Breast 66 TLLKTIIKV Brain 67 LLDVLAPLVLung, Stomach, Liver, Pancreas, Breast, Esophagus 68 YVLTQPPSVLung, Pancreas, Leukocytes, Esophagus 69 ILADLLPSLLung, Brain, Pancreas, Breast, Ovary 72 YSLEKVFGI Liver 73 GLDGIPFTVBrain, Pancreas, Breast, Melanoma 78 ALLDTVVQA Liver, Prostate 79ALADIVWRA Lung, Brain, Colon, Rectum, Liver, Pancreas,Prostate, Ovary, Esophagus 82 SLLQATDFMSLColon, Rectum, Pancreas, Esophagus 84 YLMDEGAHL Liver 85 FVMSEIRTV Liver86 GLLQGKLALL Liver, Melanoma 87 LADGVQKV Breast, Melanoma 88 TLAELHISLOvary 90 FTLEKNFVI Prostate 91 MLLSSLVSL Lung, Liver 93 GVMAGDIYSVLung, Colon, Rectum, Liver, Esophagus 94 ILHHKVYDL Liver 96 TLAETLVNLLung, Stomach, Colon, Rectum, Pancreas,Prostate, Breast, Ovary, Esophagus 97 TLISELVQALung, Colon, Rectum, Pancreas, Prostate, Breast,Melanoma, Ovary, Esophagus 98 KIPPVSPSI Lung, Liver, Breast 99 GLAPHLEQILiver, Ovary 100 KLNVAPLAV Lung, Brain, Colon, Rectum, Liver, Pancreas,Melanoma, Ovary 101 HIYDKAFITV Liver, Ovary 102 LLFDVHTTL Lung, Brain103 KLQDGLLHI Brain, Colon, Rectum, Liver 104 ALFEGVVRQILiver, Melanoma, Esophagus 105 ALADLDELLIRA Pancreas, Melanoma 107VLMDLKALLL Prostate, Leukocytes, Ovary 108 VLISVLQAI Leukocytes 109YLWSRVEKL Pancreas, Ovary 110 LLDLHSYLL Stomach, Leukocytes 111TLLETEMLL Pancreas, Breast 112 LLFDHLEPIEL Leukocytes 113 SLFDWNVKLLiver, Prostate 114 ALAVNISAA Lung, Brain, Liver, Pancreas, Esophagus115 LLDPKTIFL Colon, Rectum, Liver 116 GLVDIMVHL Ovary 117 VLFGELPALLung, Pancreas, Breast, Ovary 119 RLHDENILLLung, Brain, Colon, Rectum, Liver, Pancreas, Prostate, Ovary, Esophagus121 ALLRTVVSV Lung, Liver, Pancreas, Breast, Ovary 122 SLDPSSPQV Liver123 YVDPVITSI Lung 124 ILSPLSVAL Liver, Pancreas 125 KLDPTKTTL Prostate126 KIQEILTQV Lung, Brain, Stomach, Colon, Rectum, Liver,Pancreas, Leukocytes, Ovary, Esophagus 127 VLAPLFVYLLung, Pancreas, Breast, Melanoma 128 YLEEDVYQL Lung, Pancreas 129VLAPRVLRA Lung, Brain, Colon, Rectum, Liver, Pancreas, Ovary 130ALPTVLVGV Lung, Brain, Stomach, Colon, Rectum, Liver,Melanoma, Esophagus 131 VMAGDIYSV Lung, Liver, Pancreas, Esophagus 132SVASTITGV Liver, Breast 133 QLIDYERQLLung, Colon, Rectum, Liver, Pancreas, Esophagus 134 VADKIHSVStomach, Pancreas, Esophagus 135 VVDEGPTGVLung, Brain, Stomach, Liver, Pancreas,Leukocytes, Breast, Ovary, Esophagus 136 YQDPHSTAV Brain, Liver 137TLVAIVVGV Lung, Brain, Stomach, Colon, Rectum, Liver,Pancreas, Prostate, Breast, Ovary 138 SLDTLMTYVLung, Brain, Colon, Rectum, Pancreas, Prostate, Leukocytes, Esophagus139 ILNVDGLIGV Brain, Colon, Rectum, Liver, Prostate 140 SLANNVTSVLung, Brain, Pancreas, Melanoma, Ovary, Esophagus 141 LLVDDSFLHTVBrain, Liver, Pancreas, Melanoma, Ovary, Esophagus 142 SVDVSPPKVLung, Pancreas, Melanoma, Esophagus 143 ALFVRLLALALung, Brain, Stomach, Colon, Rectum, Liver, Melanoma, Esophagus 144RLLDVLAPLV Liver 145 SLHFLILYVLung, Brain, Colon, Rectum, Liver, Melanoma, Ovary 146 KLIDLSQVMYLLung, Colon, Rectum, Pancreas, Ovary 147 ALADKELLPSVLung, Colon, Rectum, Liver, Pancreas, Melanoma, Ovary 148 KLLTEVHAALung, Stomach, Colon, Rectum, Liver, Pancreas, Breast, Ovary, Esophagus149 SILTIEDGIFEV Lung, Brain, Colon, Rectum, Liver, Pancreas,Prostate, Leukocytes, Breast, Melanoma, Ovary 150 TLMPNINKL Liver

TABLE 4BPeptides according to the present invention and their specific uses in otherproliferative diseases, especially in other cancerous diseases. The table shows, like Table4A, for selected peptides on which additional tumor types they were found showing over-presentation (including specific presentation) on more than 5% of the measured tumorsamples, or presentation on more than 5% of the measured tumor samples with a ratioof geometric means tumor vs normal tissues being larger than 3. Over-presentation isdefined as higher presentation on the tumor sample as compared to the normal samplewith highest presentation. Normal tissues against which over-presentation was testedwere: adipose tissue, adrenal gland, blood cells, blood vessel, bone marrow, brain,cartilage, esophagus, eye, gallbladder, heart, kidney, large intestine, liver, lung, lymphnode, nerve, pancreas, parathyroid gland, peritoneum, pituitary, pleura, salivary gland,skeletal muscle, skin, small intestine, spleen, stomach, thymus, thyroid gland, trachea,ureter, urinary bladder. SEQ ID No Sequence Additional Entities 2ILWREVVTL Melanoa, NHL 4 FLSQDIITV Uterine Cancer 6 VLFELSKTV AML 8GLASFKSFL Melanoma, Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer 10 KLLQNNYGLBRCA, Gallbladder Cancer, Bile Duct Cancer 12 ALDPSGNQLI Brain Cancer 13KIMAQILTV CLL, NHL 14 ALLTETIFL NHL 15 ILIKHLVKV Uterine Cancer, NHL 17ILAQQVHAL Uterine Cancer 18 YVLDLAAKVSCLC, CLL, BRCA, Melanoma, Uterine Cancer, NHL 20 AVANTTFTVEsophageal Cancer, Urinary bladder cancer, Uterine Cancer 21 RLIQGDQILSVSCLC, BRCA, Esophageal Cancer, Urinary bladder cancer,Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, OC 24 FTLGTTVFLMelanoma, Uterine Cancer, NHL 26 SLMEILYTLMelanoma, Gallbladder Cancer, Bile Duct Cancer, AML, OC, SCLC 27SLSDLLVSL CRC, Melanoma, Uterine Cancer, AML, NHL 28 FIADLVVGLSCLC, CLL, AML, NHL 29 ILLDLEQALMelanoma, Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer, AML, NHL, OC 30 QLFYTKIFLMelanoma, Esophageal Cancer, Urinary bladder cancer,Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 33 FIADVVEKISCLC, BRCA, Esophageal Cancer 34 ELNNQNFYLCRC, Urinary bladder cancer, AML, NHL 36 SLFGKKYILUrinary bladder cancer, Uterine Cancer, GallbladderCancer, Bile Duct Cancer 38 VLLDTILQLNSCLC, SCLC, CLL, BRCA, Esophageal Cancer, Urinarybladder cancer, Uterine Cancer, Gallbladder Cancer, BileDuct Cancer, AML, NHL, OC 39 YLLNLNHLGL NHL 40 YIQEHLLQI Melanoma 43YLKDELDEL Esophageal Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL45 ALLGIPLTL BRCA, Melanoma, Urinary bladder cancer, Uterine Cancer, OC46 GLSEVLVQI SCLC, HCC, Melanoma, Urinary bladder cancer,Gallbladder Cancer, Bile Duct Cancer, NHL 48 VVASNIMEV BRCA, Melanoma 49VLIVEVPGV Gallbladder Cancer, Bile Duct Cancer, OC 50 SLSDHIVLLPC, Melanoma, Esophageal Cancer, Urinary bladdercancer, Gallbladder Cancer, Bile Duct Cancer 51 NLWPMILTLMelanoma, Uterine Cancer, Gallbladder Cancer, Bile DuctCancer, Esophageal Cancer 52 SILDAVQRVSCLC, BRCA, Melanoma, Uterine Cancer 54 ALVAKGLVQA NHL 55 YLALILPVL CLL56 ILMDFSNSM Urinary bladder cancer, Gallbladder Cancer, Bile DuctCancer 57 SLQKEILYL Melanoma 59 SLKNNVVSVMelanoma, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 61SLMGILLRI BRCA 62 VLAGPAFLVQL Uterine Cancer 63 GLIEDHFDVTV SCLC 64LLAASVALA Brain Cancer, AML 65 IIYGGSVTGACLL, Melanoma, Urinary bladder cancer, Uterine Cancer 66 TLLKTIIKVMelanoma, Urinary bladder cancer 67 LLDVLAPLVCRC, Melanoma, Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer 68 YVLTQPPSV BRCA 69 ILADLLPSLSCLC, Melanoma, Esophageal Cancer, Uterine Cancer, AML, NHL 72 YSLEKVFGIBrain Cancer, GC, CRC, PrC, BRCA, Melanoma, OC,Urinary bladder cancer, AML, PC 73 GLDGIPFTVGC, Urinary bladder cancer, Uterine Cancer, AML 75 FLIKSINLVCLL, Melanoma, AML 76 VLADDHLIEVHCC, Gallbladder Cancer, Bile Duct Cancer 77 SLIKHKIMLCRC, Melanoma, Uterine Cancer 78 ALLDTVVQA BRCA 79 ALADIVWRASCLC, BRCA, Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer, Esophageal Cancer, OC 80 KLASMLETLMelanoma 81 SLLPALPKL BRCA 82 SLLQATDFMSLNSCLC, Brain Cancer, BRCA, Melanoma, Uterine Cancer, NHL, PC 83IQWSIVPEV CLL, Melanoma, Gallbladder Cancer, Bile Duct Cancer, AML, NHL84 YLMDEGAHL Gallbladder Cancer, Bile Duct Cancer 85 FVMSEIRTVUterine Cancer, Gallbladder Cancer, Bile Duct Cancer 86 GLLQGKLALLUterine Cancer, NHL 88 TLAELHISL CLL, Esophageal Cancer 89 SLLLAVTEVSCLC, BRCA, Melanoma, Urinary bladder cancer 90 FTLEKNFVI CRC, BRCA, PC91 MLLSSLVSL SCLC, Melanoma, Urinary bladder cancer, GallbladderCancer, Bile Duct Cancer 92 FLFRDILEL Melanoma, AML 93 GVMAGDIYSVGallbladder Cancer, Bile Duct Cancer, PC 95 KLTDVGIATL Melanoma, NHL 96TLAETLVNL SCLC, HCC, CLL, Melanoma, Urinary bladder cancer,Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, AML, NHL 97TLISELVQA SCLC, GC, HCC, CLL, Urinary bladder cancer, UterineCancer, Gallbladder Cancer, Bile Duct Cancer, NHL 98 KIPPVSPSIUterine Cancer, NHL 99 GLAPHLEQI BRCA, Melanoma, AML, NHL 100 KLNVAPLAVCLL, BRCA, Uterine Cancer, AML, NHL, OC 101 HIYDKAFITV SCLC, BRCA 102LLFDVHTTL SCLC, Melanoma, Urinary bladder cancer, Uterine Cancer, AML103 KLQDGLLHI SCLC, PC, Melanoma, OC, NHL 104 ALFEGVVRQIUrinary bladder cancer 105 ALADLDELLIRA BRCA 106 VLMDLKALL AML 107VLMDLKALLL AML 108 VLISVLQAI BRCA, AML, NHL 109 YLWSRVEKL CLL, NHL 110LLDLHSYLL Uterine Cancer 111 TLLETEMLL Urinary bladder cancer 112LLFDHLEPIEL BRCA, NHL 113 SLFDWNVKLSCLC, BRCA, Esophageal Cancer, Gallbladder Cancer,Bile Duct Cancer, AML, NHL 114 ALAVNISAAGC, CRC, CLL, BRCA, Melanoma, Urinary bladder cancer,Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, AML, NHL, PC NSCLC= non-small cell lung cancer, SCLC = small cell lung cancer, RCC =kidney cancer, CRC = colon or rectum cancer, GC = stomach cancer, HCC =liver cancer, PC = pancreatic cancer, PrC = prostate cancer, BRCA =breast cancer, MCC = Merkel cell carcinoma, OC = ovarian cancer, NHL =non-Hodgkin lymphoma, AML = acute myeloid leukemia, CLL = chroniclymphocytic leukemia.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 21, 27, 29, 30, 33, 37, 39, 44, 50, 51, 52, 63, 67,68, 69, 79, 91, 93, 96, 97, 98, 100, 102, 114, 117, 119, 121, 123, 126,127, 128, 129, 130, 131, 165, 137, 138, 140, 142, 143, 145, 146, 147,148 and 149, for the—in one preferred embodiment combined—treatment oflung cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No 31, 38, 52, 66, 69, 73, 79, 100, 102, 103, 114, 119,126, 129, 130, 135, 136, 137, 138, 139, 140, 141, 143, 145 and 149 forthe—in one preferred embodiment combined—treatment of brain cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of Seq ID No 32, 67, 96, 110, 126, 130, 134, 135, 137, 143 and 148for the—in one preferred embodiment combined—treatment of gastriccancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of Seq ID No 21, 26, 30, 36, 37, 46, 63, 79, 82, 93, 96, 97, 100,103, 115, 119, 126, 129, 130, 133, 137, 138, 139, 143, 145, 146 147, 148and 149 for the—in one preferred embodiment combined—treatment ofcolorectal cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of Seq ID No 1, 5, 15, 21, 23, 24, 27, 38, 39, 47, 67, 72, 78, 79,84, 85, 86, 91, 93, 94, 98, 99, 100, 101, 103, 104, 113, 114, 115, 119,121, 122, 124, 126, 129, 130, 131, 132, 133, 135, 136, 137, 139, 141,143, 144, 145, 147, 148, 149 and 150 for the—in one preferred embodimentcombined—treatment of hepatic cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of Seq ID No 8, 16, 26, 27, 29, 37, 38, 51, 52, 56, 59, 67, 68, 69,73, 79, 82, 96, 97, 100, 105, 109, 111, 114, 117, 119, 121, 124, 126,127, 128, 129, 131, 133, 134, 135, 137, 138, 140, 141, 142, 146, 147,148 and 149 for the—in one preferred embodiment combined—treatment ofpancreatic cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of Seq ID No 24, 26, 29, 45, 63, 78, 79, 90, 96, 97, 107, 113, 119,125, 137, 138, 139 and 149 for the—in one preferred embodimentcombined—treatment of prostate cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of Seq ID No 14, 24, 27, 33, 39, 61, 63, 68, 107, 108, 110, 112,126, 135, 138 and 149 for the—in one preferred embodimentcombined—treatment of leukemias.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of Seq ID No 16, 29, 65, 67, 69, 73, 87, 96, 97, 98, 111, 117, 121,127, 132, 135, 137, 148 and 149 for the—in one preferred embodimentcombined—treatment of breast cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of Seq ID No 28, 34, 38, 44, 47, 54, 55, 63, 73, 86, 87, 97, 100,104, 105, 127, 130, 140, 141, 142, 143, 145, 147 and 149 for the—in onepreferred embodiment combined—treatment of melanoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of Seq ID No 2, 12, 16, 21, 26, 29, 30, 31, 33, 37, 38, 44, 45, 49,52, 54, 59, 63, 69, 79, 88, 96, 97, 99, 100, 101, 107, 109, 116, 117,119, 121, 126, 129, 135, 137, 140, 141, 145, 146, 147, 148 and 149 forthe—in one preferred embodiment combined—treatment of ovarian cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of Seq ID No 24, 34, 45, 51, 59, 67, 68, 79, 82, 93, 96, 97, 104,114, 119, 126, 130, 131, 133, 134, 135, 138, 140, 141, 142, 143 and 148for the—in one preferred embodiment combined—treatment of esophagealcancer.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofRCC, lung cancer, brain cancer, gastric cancer, colorectal cancer,hepatic cancer, pancreatic cancer, prostate cancer, leukemias, breastcancer, melanoma, ovarian cancer, and esophageal cancer, and preferablyRCC.

Preferably, the present invention relates to the use of the peptidesaccording to the present invention according to SEQ ID NO: 1 and/or 15for the—preferably combined—treatment of a proliferative diseaseselected from the group of RCC, lung cancer, brain cancer, gastriccancer, colorectal cancer, hepatic cancer, pancreatic cancer, prostatecancer, leukemias, breast cancer, melanoma, ovarian cancer, andesophageal cancer, and preferably RCC.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 114.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to SEQ ID 1 to SEQ ID151 according to the invention at hand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 114, preferably containing SEQ IDNo. 1 to SEQ ID No. 63, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is for a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are RCC, lung cancer, brain cancer,stomach cancer, colon or rectal cancer, liver cancer, pancreatic cancer,prostate cancer, leukemias, breast cancer, melanoma, ovarian cancer, andesophageal cancer, and preferably RCC cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”that can be used in the diagnosis of cancer, preferably RCC. The markercan be over-presentation of the peptide(s) themselves, orover-expression of the corresponding gene(s). The markers may also beused to predict the probability of success of a treatment, preferably animmunotherapy, and most preferred an immunotherapy targeting the sametarget that is identified by the biomarker. For example, an antibody orsoluble TCR can be used to stain sections of the tumor to detect thepresence of a peptide of interest in complex with MHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

ABCC3 is associated with hepatocellular carcinoma, ovarian cancer,rectal cancer, osteosarcoma, breast cancer, non-small cell lung cancer,glioblastoma multiforme and pancreatic ductal adenocarcinoma(Mohelnikova-Duchonova et al., 2013; Molina-Pinelo et al., 2014; Goodeet al., 2014; Liu et al., 2014c; Yu et al., 2014; Sedlakova et al.,2015; Zuniga-Garcia et al., 2015; Wang et al., 2014c).

ACLY is aberrantly expressed in various tumors, such as breast, liver,colon, lung and prostate cancers, and is correlated reversely with tumorstage and differentiation (Zu et al., 2012).

ADAM8 over-expression in pancreatic cancer is associated with increasedmigration and invasiveness of pancreatic ductal adenocarcinoma cells(Schlomann et al., 2015). ADAM8 is involved in tumor cell migration andinvasion in lung cancer, renal cell carcinoma and brain cancers(Mochizuki and Okada, 2007).

ADCY5 gene hyper-methylation and reduced mRNA expression occurs in acutelymphoblastic leukemia, chronic lymphocytic leukemia and lungadenocarcinoma (Kuang et al., 2008; Tong et al., 2010; Sato et al.,2013a).

AHNAK2 is an important element of the non-classical secretion pathway offibroblast growth factor 1 (FGF1), a factor involved in tumor growth andinvasion (Kirov et al., 2015).

The expression of AIFM2 was shown to be down-regulated in the majorityof human tumors (Wu et al., 2004; Mei et al., 2006). AIFM2 wasidentified as one of nine genes which were associated with functionalsuppression of tumorigenicity in ovarian cancer cell lines (Notaridou etal., 2011).

AKR1A1 was shown to be up-regulated in breast cancer and is associatedwith lung cancer and laryngeal cancer (Penning, 2014; Hlavac et al.,2014; Kim et al., 2012).

ALPK2 expression is down-regulated in colorectal adenoma and plays apossible role in the transition of normal colonic crypt to adenoma(Yoshida et al., 2012). ALPK2 shows a strong association between copynumber loss and under-expression in gastric cancer (Junnila et al.,2010).

ANGPTL4 was shown to be up-regulated in breast cancer, serous ovariancancer and is associated with glioblastoma, hepatocellular carcinoma,oral squamous cell carcinoma and lung cancer (Ferguson et al., 2013;Tanaka et al., 2015; Ng et al., 2014; Garner et al., 2015; Schumann etal., 2015; Johnson et al., 2015).

APOL1 expression is down-regulated in renal cell carcinoma tissues andcell lines (Hu et al., 2012).

ARRDC3 is associated with breast cancer and prostate cancer (Wang etal., 2014a; Huang et al., 2012a).

ATG2B frameshift mutations are common in gastric and colon carcinomaswith high microsatellite instability (Kang et al., 2009).

ATP11A was shown to be up-regulated in colorectal cancer and isassociated with lymphoblastic leukemia and is suggested as a biomarkerof metastasis in colorectal cancer (Miyoshi et al., 2010; Zhang et al.,2005).

ATP2A1 was shown to be up-regulated in cancer cachexia (Fontes-Oliveiraet al., 2013).

ATP2A2 is associated with skin cancer, colon cancer and lung cancer(Korosec et al., 2006; Hovnanian, 2007).

CACNA1H is associated with aldosterone-producing adenomas, prostatecancer and breast cancer (Felizola et al., 2014; Asaga et al., 2006;Gackiere et al., 2013).

CACNA1I is associated with colon cancer, breast cancer and prostatecancer (Basson et al., 2015).

CARD8 is highly expressed in several cancer cell lines includingovarian, breast and lung cancer as well as in tissues derived frompatients with colorectal carcinoma, gastric or breast cancer (Pathan etal., 2001; Yamamoto et al., 2005).

CCT6A is associated with testicular germ cell tumors and malignantmelanomas (Tanic et al., 2006; Alagaratnam et al., 2011).

CIT is frequently up-regulated in hepatocellular carcinoma (HCC) ascompared with adjacent non-tumor tissues. CIT knock-down by RNAisuppresses tumorigenicity of HCC cells in vivo (Fu et al., 2011).

CLIC4 down-regulation in tumor cells and up-regulation in tumor stromais common to many human cancers including renal, ovarian, breast, lungand cutaneous cancers, and marks malignant progression (Suh et al.,2007a; Okudela et al., 2014; Suh et al., 2007b).

Differential expression of COL18A1 was reported for bladder cancer,rhabdoid tumors and ovarian carcinoma and specific polymorphisms withinthe gene were shown to increase the risk for sporadic breast cancer(Fang et al., 2013; Gadd et al., 2010; Lourenco et al., 2006; Peters etal., 2005).

COL6A2 is associated with cervical cancer, poor overall survival inhigh-grade serous ovarian cancer, B-precursor acute lymphoblasticleukemia, hepatocellular carcinoma, primary and metastatic brain tumors,squamous cell carcinoma of the lung, head and neck squamous cellcarcinoma and was described as a potential DNA methylation for cervicalcancer (Cheon et al., 2014; Chen et al., 2014c; Vachani et al., 2007;Liu et al., 2010b; Seong et al., 2012; Hogan et al., 2011).

CUL7 is associated with pancreatic cancer and hepatocellular carcinoma(Wang et al., 2014b; Paradis et al., 2013).

CYB5A expression is down-regulated in hepatocellular carcinoma (Khan etal., 2013a). CYB5A is a prognostic factor for pancreatic ductaladenocarcinoma that exerts its tumor-suppressor function throughautophagy induction and TRAF6 modulation (Giovannetti et al., 2014).CYB5A encodes an enzyme which detoxifies carcinogenic molecules and is aprognostic factor for pancreatic cancer (Blanke et al., 2014;Giovannetti et al., 2014).

CYFIP2 expression is increased in newly formed lymph nodes in breastcancer (Gantsev et al., 2013). CYFIP2 expression is reduced in humangastric tumor samples, compared with control tissues (Cheng et al.,2013). CYFIP2 is one of several apoptosis-related genes methylated inchronic lymphocytic leukemia (Halldorsdottir et al., 2012).

CYP2J2 is an enzyme, which was shown to be over-expressed in a varietyof human cancers, including esophageal, lung, breast, stomach, liver andcolon cancer. CYP2J2 increases the proliferation and inhibits theapoptosis of carcinoma cells by promoting EGFR phosphorylation andactivation of PI3K and MAPK signaling and further metabolizes tyrosinekinase inhibitors, thereby conferring resistance to anti-cancer agents(Jiang et al., 2005; Narjoz et al., 2014).

DCBLD2 is up-regulated in glioblastomas and head and neck cancers (HNCs)and is required for EGFR-stimulated tumorigenesis (Feng et al., 2014).Furthermore, DCBLD2 is up-regulated in highly metastatic lung cancersublines and tissue samples (Koshikawa et al., 2002). In contrast, theexpression of DCBLD2 is silenced by hypermethylation of its promoter ingastric cancer (Kim et al., 2008).

DDX41 is associated with acute myeloid leukemia (Antony-Debre andSteidl, 2015).

DROSHA, one of the two critical enzymes in microRNA biosynthesis, isover-expressed in a number of cancers including gastrointestinal tumors,breast cancer and cervical cancer and appears to enhance proliferation,colony formation and migration of tumor cells (Avery-Kiejda et al.,2014; Havens et al., 2014; Zhou et al., 2013).

Single nucleotide polymorphisms in the DUSP14 gene are associated withaltered melanoma risk (Yang et al., 2014a; Liu et al., 2013).

EGFR was shown to be up-regulated in breast cancer and salivary glandadenoid carcinoma and is associated with non-small cell lung cancer,hepatocellular carcinoma and colorectal cancer (Dienstmann et al., 2015;Wang et al., 2015b; Steinway et al., 2015; Xiao et al., 2015; Inoue etal., 2015).

EGLN3 expression was shown to be up-regulated in non-small cell lungcancer and renal cell carcinomas. Moreover, EGLN3 is associated withclear-cell renal cell carcinoma and colorectal cancer (Tanaka et al.,2014; Yang et al., 2014d; Toth et al., 2014; Chu et al., 2014).

ENO1 expression was shown to be up-regulated in non-small cell lungcancer. Furthermore, ENO1 is associated with endometrial carcinoma,pancreatic ductal adenocarcinoma, glioblastoma and nasopharyngealcarcinoma (Yang et al., 2014b; Naryzhnyi et al., 2014; Principe et al.,2015; Fu et al., 2015; Zhao et al., 2015a).

ENO2 is associated with lung cancer, solid neuroendocrine carcinoma,granular cell tumors and pancreatic cancer (Sigari et al., 2014;Zizi-Sermpetzoglou et al., 2014; Liu et al., 2014a; Bedir et al., 2015;Wang et al., 2013b).

ENO3 is associated with B cell lymphoma, alveolar soft part sarcoma,rhabdomyosarcoma and neuroblastoma (Oka et al., 1989; Mukai et al.,1986; Royds et al., 1985; Ishiguro et al., 1984).

ENPP3 is associated with neuroblastoma, stage II colorectal cancer, headand neck squamous cell carcinoma, acute basophilic leukemia and bileduct carcinoma (Agesen et al., 2012; Staal-Viliare et al., 2007;Gomez-Villafuertes et al., 2014; Yano et al., 2004; Thiel et al., 2011).

ERAP1 is associated with cervical carcinoma, renal cell carcinoma,esophageal squamous cell carcinoma, melanoma, ovarian carcinoma andneuroblastoma (Mehta et al., 2015; Forloni et al., 2010; Liu et al.,2010a; Kamphausen et al., 2010; Ayshamgul et al., 2011; Stoehr et al.,2013).

ESM1 expression was shown to be elevated in gastric cancer and isassociated with hepatoblastoma, nasopharyngeal carcinoma and ovariancancer and may be a potential biomarker for gastric cancer (Yu et al.,2013; Dong et al., 2014; Lv et al., 2014; El Behery et al., 2013).

FHL2 was shown to be up-regulated in acute myeloid leukemia, ovariancancer, lung cancer, colon carcinoma, breast cancer, pancreatic ductaladenocarcinoma, and human malignant melanoma and down-regulated inprostate cancer and rhabdomyosarcoma (Kleiber et al., 2007; Westphal etal., 2015; Qian et al., 2010; Zienert et al., 2015).

FKBP10 was identified as a novel gene that participates in theacquisition and maintenance of the Adriamycin-resistant phenotype inleukemia cells (Sun et al., 2014b).

FKBP10 has been associated with colorectal cancer through itsup-regulation (Olesen et al., 2005). In contrast, the under-expressionof FKBP10 was characteristic for epithelial ovarian carcinomas (Quinn etal., 2013).

FLT1 is associated with colon cancer, prostate cancer, non-small celllung cancer and pancreatic cancer (Awasthi et al., 2015; Heist et al.,2015; Tsourlakis et al., 2015; Zhang et al., 2015).

FZD1 is associated with esophageal cancer, thyroid carcinoma, uterussarcoma, prostate cancer, squamous cell/adenosquamous carcinoma andadenocarcinoma of the gallbladder, colon cancer and breast cancer(Goksel et al., 2014; Hung et al., 2014; Davidov et al., 2014; Su etal., 2015; Zhang et al., 2012a; Planutis et al., 2013; Devaney et al.,2013; Li et al., 2014a).

FZD2 was shown to be up-regulated in esophageal cancer and is associatedwith gastrointestinal stromal tumor, salivary adenoid cystic carcinomaand colorectal cancer (Wang and Zheng, 2014; Ding et al., 2015; Prakashet al., 2005; Liu et al., 2014b).

FZD7 was shown to be up-regulated in ovarian cancer and is associatedwith cervical cancer, hepatocellular carcinoma, colorectal cancer,melanoma, breast cancer, gastric cancer and central neurocytoma (Anastaset al., 2014; Li et al., 2014c; Gonzalez et al., 2014; Song et al.,2014; Deng et al., 2015; Asad et al., 2014; Vasiljevic et al., 2013;Rocken and Warneke, 2012; Dey et al., 2013).

GAL3ST1 activity is increased in renal cell carcinoma (RCC) tissue andthe RCC cell line SMKT-R3 (Honke et al., 1996). GAL3ST1 expression isup-regulated in ovarian epithelial carcinoma cells versus normal ovarianstromal tissue and normal surface ovarian epithelial cells (Liu et al.,2010c).

GALNT2, the N-acetylgalactosaminyltransferase 2, was shown to exertanti-proliferative and anti-metastatic activity through the decrease ofMMP-2 and TGF-β1 in gastric cancer cells and through the inhibition EGFreceptor activity in hepatocellular carcinoma, where it is frequentlydown-regulated. In contrast, in squamous cell carcinoma over-expressionof GALNT2 was reported to enhance the invasive potential of tumor cellsby modifying 0-glycosylation and EGFR activity (Hua et al., 2012; Lin etal., 2014; Wu et al., 2011).

GRAMD4 is up-regulated in hepatocellular carcinoma (HCC) cell lines andHCC tissues, and the increased expression is correlated with theclinicopathological characteristics of HCC (Zhang et al., 2013).

HAVCR1 was described as a novel biomarker candidate associated withovarian clear cell carcinoma and renal cell carcinoma (Bonventre, 2014;Kobayashi et al., 2015). HAVCR1 was shown to activate theIL-6/STAT-3/HIF-1A axis in clear cell renal cell carcinoma-derived celllines and determines tumor progression and patient outcome (Cuadros etal., 2014). Constitutive expression of HAVCR1 in the kidney wasdescribed as a potential susceptibility trait for clear cell renal cellcarcinoma development (Cuadros et al., 2013). HAVCR1 was described asbeing up-regulated in renal cell and ovarian clear cell carcinomas andcolorectal cancer (Wang et al., 2013c). HAVCR1up-regulation wasdescribed as a potential diagnostic biomarker for colorectal cancer anda prognostic marker for a longer disease-free interval after surgery,which may also be involved in the metastatic cascade in colorectalcancer (Wang et al., 2013c). HAVCR1 was shown to be associated with Tcell large granular lymphocyte leukemia (Wlodarski et al., 2008).

HSF2BP encodes the HSF2 binding protein which associates with HSF2 andmay be involved in modulating HSF2 activation (RefSeq, 2002).

HSF4 encodes heat-shock transcription factor 4, which activatesheat-shock response genes under conditions of heat or other stresses(RefSeq, 2002). HSF4 was shown to be down-regulated in glioblastoma(Mustafa et al., 2010).

Different studies suggest an important role of HSPA2 in diseaseprogression of cervical cancer, renal cell carcinoma and bladder cancer.Polymorphisms within the gene are associated with the development ofgastric cancer (Ferrer-Ferrer et al., 2013; Garg et al., 2010a; Garg etal., 2010b; Singh and Suri, 2014).

HSPA8 was shown to be over-expressed in esophageal squamous cellcarcinoma and high expression levels of HSPA8 in esophageal cancer cellsin vitro counter-acted oxidative stress-induced apoptosis of thesecells. Furthermore, HSPA8 is over-expressed in multiple myeloma andcolonic carcinoma and BCR-ABL1-induced expression of HSPA8 promotes cellsurvival in chronic myeloid leukemia (Chatterjee et al., 2013; Dadkhahet al., 2013; Jose-Eneriz et al., 2008; Kubota et al., 2010; Wang etal., 2013a).

HSPG2 is associated with melanoma, oral squamous cell carcinoma, clearcell renal cell carcinoma and prostate tumor and its down-regulation wasshown in hepatocellular carcinoma and colon tumor (Nikitovic et al.,2014; Warren et al., 2014; Gbormittah et al., 2014; Zaghloul et al.,2015; Kawahara et al., 2014; Suhovskih et al., 2015; 2015; Elewa et al.,2015; Lai et al., 2011).

HTRA1 is associated with hepatocellular carcinoma, splenic marginal zonelymphoma, squamous cell carcinoma and neuroblastoma and was shown to bedown-regulated in gastric cancer, breast cancer, gallbladder cancer andlung adenocarcinoma (Xu et al., 2014; D'Angelo et al., 2014; Fujinaga etal., 2014; Sahasrabuddhe et al., 2014; Franco et al., 2015; Arribas etal., 2015; Zhao et al., 2015b; Bao et al., 2015).

HTRA3 was shown to be up-regulated in oral squamous cell carcinoma andthyroid carcinoma and down-regulated in ovarian cancer, breast cancer,endometrial cancer and lung cancer, and is associated with colorectalcancer and may be a potential prognostic biomarker for oral cancer(Karagiannis et al., 2014; Zhao et al., 2014; Moriya et al., 2015;Narkiewicz et al., 2009; Beleford et al., 2010; Zurawa-Janicka et al.,2012; Yin et al., 2013).

IGF2BP3 encodes insulin-like growth factor II mRNA binding protein 3, anoncofetal protein, which represses translation of insulin-like growthfactor II (RefSeq, 2002). In vitro studies have shown that IGF2BP3promotes tumor cell proliferation, adhesion, and invasion. Furthermore,IGF2BP3 has been shown to be associated with aggressive and advancedcancers (Bell et al., 2013; Gong et al., 2014). IGF2BP3 over-expressionhas been described in numerous tumor types and correlated with poorprognosis, advanced tumor stage and metastasis, as for example inneuroblastoma, colorectal carcinoma, intrahepatic cholangiocarcinoma,hepatocellular carcinoma, prostate cancer, and renal cell carcinoma(Bell et al., 2013; Findeis-Hosey and Xu, 2012; Hu et al., 2014; Szarvaset al., 2014; Jeng et al., 2009; Chen et al., 2011; Chen et al., 2013;Hoffmann et al., 2008; Lin et al., 2013; Yuan et al., 2009).

IGLC1 newly acquired and recurrent deletions were detected in 3 out of45 patients that showed tyrosine kinase inhibitor (TKI) resistance inchronic myeloid leukemia (CML) (Nowak et al., 2010).

IGLC2 loss of heterozygosity has been shown in 50% of informative casesof intracranial meningiomas (Kim et al., 1993).

IGLJ3 is the joining gene universally used for the composition of thepreferred light chain IG lambda in hairy cell leukemia (Forconi et al.,2008).

IGLV2-14 is the third most frequent IGLV gene in chronic lymphocyticleukemia (CLL) compared with the relevant repertoires from normal,auto-reactive, and neoplastic cells (Stamatopoulos et al., 2005).

IGLV3-21 is the most frequent IGLV gene in chronic lymphocytic leukemia(CLL) compared with the relevant repertoires from normal, auto-reactive,and neoplastic cells (Stamatopoulos et al., 2005).

INADL is down-regulated in non-small cell lung cancer in response tocisplatin-gemcitabine combination chemotherapy (Ma et al., 2015).

ITGA3 was shown to be up-regulated in colorectal carcinoma and isassociated with prostate cancer, epidermoid carcinoma, early gastriccarcinoma and osteosarcoma (Yang et al., 2014c; Chong et al., 2014;Lustosa et al., 2014; Ren et al., 2014; Bauer et al., 2014;Mertens-Walker et al., 2015).

ITGB4 is associated with prostate cancer, gastric cancer, breast cancer,oral squamous cell carcinoma and ovarian cancer and was shown to beup-regulated in pancreatic ductal adenocarcinoma (Chen et al., 2014b;Xin et al., 2014; Zubor et al., 2015; Masugi et al., 2015; Gao et al.,2015b; Kawakami et al., 2015). ITGB4 (also called CD104) tends toassociate with the alpha 6 subunit and is likely to play a pivotal rolein the biology of several invasive carcinomas such as esophagealsquamous cell carcinoma, bladder and ovarian carcinoma (Kwon et al.,2013; Pereira et al., 2014; Chen et al., 2014b). A single nucleotidepolymorphism in ITGB4 seems to influence tumor aggressiveness andsurvival and may have prognostic value for breast cancer patients(Brendle et al., 2008).

IVNS1ABP is associated with BCL1/JH positive multiple myelomas and mightbe a potential prognostic marker of multiple myelomas (Ni et al., 2012).

JAG2 was shown to be up-regulated in pancreatic ductal adenocarcinoma,hepatocellular carcinoma and retinoblastoma and is associated withmultiple myeloma, endometrial cancer, prostate cancer, osteosarcoma,head and neck cancer and urothelial carcinoma of bladder (Sun et al.,2014a; Li et al., 2014b; Carvalho et al., 2014; Xiao et al., 2014; Zhanget al., 2014a; Sasnauskiene et al., 2014; Lu et al., 2014; Hu et al.,2015; Li et al., 2013).

KCNK15 gene hyper-methylation was found in several cell lines, includingcolon cancer, leukemia, and bladder cancer (Shu et al., 2006).

KIF12 was shown to be over-expressed in breast cancer and is associatedwith kidney tumors, uterus cancer and pancreatic cancer (Katoh andKatoh, 2005; Tan et al., 2012).

KLHDC7B is associated with cervical squamous cell carcinoma and is apotential biomarker for cervical squamous cell carcinoma (Guo et al.,2015).

LRP2 is associated with hepatocellular carcinoma, pancreatic cancer,malignant melanoma, primary central nervous system lymphoma and clearcell renal cell carcinoma (Fernandez-Banet et al., 2014; Andersen etal., 2015; Pedersen et al., 2010; Schuetz et al., 2005; Anderson et al.,2013).

LRRK2 is associated with hormone-related cancer, breast cancer andconcomitant non-skin cancers in Parkinson's disease patients and may beassociated with hematological cancers in patients with Parkinson'sdisease (Ruiz-Martinez et al., 2014; Agalliu et al., 2015; Inzelberg etal., 2012).

MACC1 is over-expressed in many cancer entities including gastric,colorectal, lung and breast cancer and is associated with cancerprogression, metastasis and poor survival of patients (Huang et al.,2013; Ma et al., 2013; Stein, 2013; Wang et al., 2015a; Wang et al.,2015c; Ilm et al., 2015). MACC1 promotes carcinogenesis throughtargeting beta-catenin and PI3K/AKT signaling pathways, which leads toan increase of c-Met and beta-catenin and their downstream target genesincluding c-Myc, cyclin D1, caspase9, BAD and MMP9 (Zhen et al., 2014;Yao et al., 2015).

MAGED2 over-expression is associated with melanoma, breast cancer andcolon cancer (Li et al., 2004; Strekalova et al., 2015).

MET was shown to be up-regulated in dedifferentiated liposarcoma and isassociated with melanocytic tumors, hepatocellular carcinoma, non-smallcell lung cancer, hereditary papillary kidney cancers and gastricadenocarcinomas (Petrini, 2015; Finocchiaro et al., 2015; Steinway etal., 2015; Bill et al., 2015; Yeh et al., 2015).

NAT8 is associated with lymphoblastic leukemia (Mak et al., 2014).

NDRG1 is a metastasis suppressor in cancers such as pancreatic cancerand was shown to be down-regulated in prostate cancer and colon cancer,whereas it was shown to be up-regulated in hepatocellular carcinoma andcervical adenocarcinoma (Nishio et al., 2008; Cheng et al., 2011; Bae etal., 2013; Richardson et al., 2013).

NLRC5 was shown to be down-regulated in lymphoid-derived tumor celllines (Staehli et al., 2012).

NPTX2 is down-regulated by promoter hyper-methylation in pancreaticcancer, Ewing sarcoma and glioblastoma (Zhang et al., 2012b; Alholle etal., 2013; Shukla et al., 2013).

NQO2 is associated with endometrial cancer, papillary thyroidmicrocarcinoma and esophageal cancer (Hevir-Kene and Rizner, 2015; Maliket al., 2012; Lee et al., 2013).

OPN3 expression is decreased in 5-fluorouracil-resistant hepatocellularcarcinoma cell lines Be17402 and HepG2 compared to5-fluorouracil-sensitive Be17402 and HepG2 cells (Jiao et al., 2012).

PDZD2 is associated with small intestinal neuroendocrine tumors andprostate cancer (Tam et al., 2006; Rehfeld et al., 2014).

Overexpression of PDZK1 may play a role in drug resistance of multiplemyeloma (RefSeq, 2002).

PFKFB3 was shown to be up-regulated in gastric cancer, colon cancer,lung cancer, breast cancer and is associated with pancreatic cancer,prostate cancer and glioblastoma (Minchenko et al., 2014; Fleischer etal., 2011; Ragnum et al., 2013).

PLCB1 is associated with primary head and neck squamous cell carcinoma,myeloid leukemia and glioblastoma multiforme (Guerrero-Preston et al.,2014; Waugh, 2014; Ramazzotti et al., 2011).

PLIN2 is involved in lipid storage and a plasma biomarker for thedetection of early-stage colorectal cancer (Matsubara et al., 2011).PLIN2 is significantly increased in patients with clear cell andpapillary renal cell carcinoma compared with controls. The preoperativeurinary concentrations of PLIN2 reflect the tumor size and stage(Morrissey et al., 2014). PLIN2 expression is significantly higher inlung adenocarcinoma specimens than in normal tissues and lung squamouscell carcinomas (Zhang et al., 2014b).

PLOD1 expression is associated with human breast cancer progression(Gilkes et al., 2013).

PRKCDBP was shown to be down-regulated in breast cancer brain metastasesand primary breast cancer. Additionally, PRKCDBP is associated withbreast cancer, colorectal cancer, endometrial cancer, lung cancer andgastric cancer (Bai et al., 2012; Moutinho et al., 2014; Li et al.,2015; Tong et al., 2012; Wikman et al., 2012).

RASGRP3 was shown to be up-regulated in glioblastoma, breast cancer andhuman melanoma and is associated with prostate cancer, hepatocellularcarcinoma and oral cancers (Nagy et al., 2014; Sowalsky et al., 2015;Lee et al., 2015; Yang et al., 2011; Bhatnagar et al., 2012;Martinez-Lopez et al., 2012).

RCN1 is localized to the plasma membrane in human endothelial andprostate cancer cell lines (RefSeq, 2002). RCN1 is over-expressed inbreast cancer (Amatschek et al., 2004).

REN is associated with renal cell carcinoma, pancreatic cancer,desmoplastic small round cell tumor and juxtaglomerular cell tumor(Elouazzani et al., 2014; Lee et al., 2014; Nakai et al., 2015; Araujoet al., 2015).

RGS5 was shown to be down-regulated in lung cancer, up-regulated inclear cell renal cell carcinoma, hepatocellular carcinoma, severallymphoma subtypes and parathyroid adenoma, and is associated withcolorectal cancer, neuroblastoma, ovarian cancer, non-small cell lungcancer and gastric cancer (Volz et al., 2015; Xu et al., 2015; Wang etal., 2010a; Dannenmann et al., 2013; Koh et al., 2011; Huang et al.,2012b; Altman et al., 2012; Kumps et al., 2013; Sethakorn and Dulin,2013; Hu et al., 2013).

RPGRIP1L suppresses anchorage-independent growth partly through themitotic checkpoint protein Mad2 and is a candidate tumor suppressor genein human hepatocellular carcinoma (Lin et al., 2009).

RRAD was shown to be down-regulated in lung cancer, ovarian cancer andnasopharyngeal carcinoma and is associated with glioblastoma multiforme,esophageal squamous cell carcinoma and hepatocellular carcinoma (Wang etal., 2014d; Yeom et al., 2014; Liu et al., 2015; Mo et al., 2012; Linand Chuang, 2012; Jin et al., 2013).

SEMA5B is up-regulated in renal cell carcinoma, but, so far, itsexpression has not been found in other cancer types or normal tissues(Hirota et al., 2006).

The plasma level of the plasmin-alpha 2-plasmin inhibitor complex wasshown to be a predictor of survival in non-small cell lung carcinoma andlow activity of alpha 2-antiplasmin has been observed in the blood ofthe patients with prostatic carcinoma (Taguchi et al., 1996; Zietek etal., 1996).

The SLC16A3 expression has been associated with poor prognosis inhepatocellular cancer patients and increased cell proliferation,migration and invasion in cell line experiments (Gao et al., 2015a). Thefunctional involvement of SLC16A3 in the tumorigenesis was shown in asubset of pancreatic cancer (Baek et al., 2014).

SLC16A4 was shown to be up-regulated in non-small lung cancer, glandadenoid carcinoma, pancreatic ductal carcinoma, oral squamous cellcarcinoma and gastric cancer and is associated with breast cancer andhepatocellular carcinoma (Baek et al., 2014; Gao et al., 2015a; Yan etal., 2014; Jensen et al., 2014; Koo and Yoon, 2015; Granja et al., 2015;Baenke et al., 2015).

High expression of organic anion transporter 2 and organic cationtransporter 2 is an independent predictor of good outcomes in patientswith metastatic colorectal cancer treated with FOLFOX-based chemotherapy(Tashiro et al., 2014). mRNA expression of SLC22A2, SLC22A11, SLC28A1,SLC28A3 and SLC29A1 was down-regulated in pancreatic tumors whencompared with non-neoplastic pancreatic tissues (Mohelnikova-Duchonovaet al., 2013a). SLC22A2 (also known as OCT2) regulates uptake ofcisplatin in proximal tubules, and inhibition of OCT2 protects againstsevere cisplatin-induced nephrotoxicity (Sprowl et al., 2013).

SLC47A1 is associated with prostate cancer (Joerger et al., 2015).

SLC6A13 expression was shown to be up-regulated in colon cancer (Tran etal., 2014b).

SMYD2 was shown to be up-regulated in esophageal squamous primarycarcinoma, breast cancer, liver cancer, gastric cancer and acutelymphoblastic leukemia (Sakamoto et al., 2014; Komatsu et al., 2015;Nguyen et al., 2015).

SYT9 is associated with cervical cancer and prostate cancer and maypresent a potential biomarker for cervical cancer (Chen et al., 2014c;Bao et al., 2011).

TGFBI expression was shown to be elevated in cholangiocarcinoma, hepaticcarcinoma, gastric carcinoma, esophageal squamous cell carcinoma andclear cell renal cell carcinoma. Furthermore, TGFBI was shown to beassociated with colorectal cancer (Lebdai et al., 2015; Ozawa et al.,2014; Zhu et al., 2015; Han et al., 2015).

THY1 is a candidate tumor suppressor gene in nasopharyngeal carcinomabearing anti-invasive activity (Lung et al., 2010).

TIMP1 protein expression is associated with poor prognosis in patientswith metastatic liver disease, in childhood acute lymphoblastic leukemiaand in breast cancer (Bunatova et al., 2012; Scrideli et al., 2010;Sieuwerts et al., 2007). TIMP1 is a potential serum marker for thedetection of pancreatic cancer (Slater et al., 2013). TIMP1 is awell-known thyroid cancer marker (Griffith et al., 2006).

TPI1 was shown to be down-regulated in osteosarcoma and is associatedwith breast cancer, esophageal squamous cell carcinoma, glioblastoma,endometrial cancer and ovarian cancer (Zamani-Ahmadmahmudi et al., 2014;Chen et al., 2014a; Yoshida et al., 2013; Khan et al., 2013b; Gao etal., 2014).

TRIP6 was shown to be up-regulated in Ewings's sarcoma, nasopharyngealcarcinoma and glioblastoma and is associated with breast cancer(Pavlikova et al., 2015; Lai et al., 2010; Fei et al., 2013; Grunewaldet al., 2013).

UBE2QL1 transcription was shown to be down-regulated in renal cellcarcinoma (Wake et al., 2013).

UGDH is associated with colorectal cancer, prostate cancer, ovarianserous adenocarcinoma, breast cancer and hepatocellular carcinoma(Lapointe and Labrie, 1999; Konno, 2001; Fan et al., 2009; Wei et al.,2009; Wang et al., 2010b).

VKORC1 polymorphisms might be associated with the risk of developingprostate cancer (Nimptsch et al., 2009). VKORC1 influences PIVKAII(protein induced by vitamin K absence) concentration, which is used forscreening for hepatocellular carcinoma (Wang et al., 2010c).

DETAILED DESCRIPTION OF THE INVENTION

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11 or 12 or longer, and incase of MHC class II peptides (elongated variants of the peptides of theinvention) they can be as long as 13, 14, 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 5 Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy-Weinberg formula F = 1 − (1 −Gf)². Combinations of A*02 or A*24 with certain HLA-DR alleles might beenriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004). Calculated phenotype from Allele Populationallele frequency A*02 Caucasian (North America)  49.1% A*02 AfricanAmerican (North America)  34.1% A*02 Asian American (North America) 43.2% A*02 Latin American (North American)  48.3% DR1 Caucasian (NorthAmerica)  19.4% DR2 Caucasian (North America)  28.2% DR3 Caucasian(North America)  20.6% DR4 Caucasian (North America)  30.7% DR5Caucasian (North America)  23.3% DR6 Caucasian (North America)  26.7%DR7 Caucasian (North America)  24.8% DR8 Caucasian (North America)  5.7%DR9 Caucasian (North America)  2.1% DR1 African (North) American 13.20%DR2 African (North) American 29.80% DR3 African (North) American 24.80%DR4 African (North) American 11.10% DR5 African (North) American 31.10%DR6 African (North) American 33.70% DR7 African (North) American 19.20%DR8 African (North) American 12.10% DR9 African (North) American  5.80%DR1 Asian (North) American  6.80% DR2 Asian (North) American 33.80% DR3Asian (North) American  9.20% DR4 Asian (North) American 28.60% DR5Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7Asian (North) American 13.40% DR8 Asian (North) American 12.70% DR9Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2Latin (North) American 21.20% DR3 Latin (North) American 15.20% DR4Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8Latin (North) American 18.60% DR9 Latin (North) American  2.10% A*24Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 SouthKorea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29%A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02. A vaccine may alsoinclude pan-binding MHC class II peptides. Therefore, the vaccine of theinvention can be used to treat cancer in patients that are A*02positive, whereas no selection for MHC class II allotypes is necessarydue to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculatedfrom www.allelefrequencies.net).

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:percent identity=100[1−(C/R)]wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and(ii) each gap in the Reference Sequence and(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and(iiii) the alignment has to start at position 1 of the alignedsequences; and R is the number of bases or amino acids in the ReferenceSequence over the length of the alignment with the Compared Sequencewith any gap created in the Reference Sequence also being counted as abase or amino acid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 114 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 114, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 114. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO: 114, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or-II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or -II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acids whose incorporation do not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 6 Variants and motif of the peptides according to SEQ ID NO: 1, 4and 5. Position 1 2 3 4 5 6 7 8 9 SEQ ID NO: 1 A L I V S L P Y L VariantV I A M V M I M M A A V A I A A A V V V I V V A T V T I T T A Q V Q I QQ A SEQ ID NO: 4 F L S Q D I I T V Variant I L A M M I M L M A A A I A LA A V V I V L V A T T I T L T A Q Q I Q L Q A SEQ ID NO: 5 Y L Y P N L TR L Variant V I A M V M I M M A A V A I A A A V V V I V V A T V T I T TA Q V Q I Q Q A

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 114.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO: 114 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an am ido group may be addedto the peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich (www.sigma-aldrich.com)provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lukas et al.(Lukas et al., 1981) and by references as cited therein. TemporaryN-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl(Fmoc) group. Repetitive cleavage of this highly base-labile protectinggroup is done using 20% piperidine in N, N-dimethylformamide. Side-chainfunctionalities may be protected as their butyl ethers (in the case ofserine threonine and tyrosine), butyl esters (in the case of glutamicacid and aspartic acid), butyloxycarbonyl derivative (in the case oflysine and histidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N,N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used include ethanedithiol, phenol, anisole andwater, the exact choice depending on the constituent amino acids of thepeptide being synthesized. Also a combination of solid phase andsolution phase methodologies for the synthesis of peptides is possible(see, for example, (Bruckdorfer et al., 2004), and the references ascited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of natural TUMAPsrecorded from RCC samples (N=18*02-positive samples) with thefragmentation patterns of corresponding synthetic reference peptides ofidentical sequences. Since the peptides were directly identified asligands of HLA molecules of primary tumors, these results provide directevidence for the natural processing and presentation of the identifiedpeptides on primary cancer tissue obtained from 18 RCC patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from RCC tissue samples were purified andHLA-associated peptides were isolated and analyzed by LC-MS (seeexamples). All TUMAPs contained in the present application wereidentified with this approach on primary RCC samples confirming theirpresentation on primary RCC.

TUMAPs identified on multiple RCC and normal tissues were quantifiedusing ion-counting of label-free LC-MS data. The method assumes thatLC-MS signal areas of a peptide correlate with its abundance in thesample. All quantitative signals of a peptide in various LC-MSexperiments were normalized based on central tendency, averaged persample and merged into a bar plot, called presentation profile. Thepresentation profile consolidates different analysis methods likeprotein database search, spectral clustering, charge state deconvolution(decharging) and retention time alignment and normalization.

Furthermore, the discovery pipeline XPRESIDENT® v2. allows for a directabsolute quantitation of MHC-, preferably HLA-restricted, peptide levelson cancer or other infected tissues. Briefly, the total cell count wascalculated from the total DNA content of the analyzed tissue sample. Thetotal peptide amount for a TUMAP in a tissue sample was measured bynanoLC-MS/MS as the ratio of the natural TUMAP and a known amount of anisotope-labelled version of the TUMAP, the so-called internal standard.The efficiency of TUMAP isolation was determined by spiking peptide:MHCcomplexes of all selected TUMAPs into the tissue lysate at the earliestpossible point of the TUMAP isolation procedure and their detection bynanoLC-MS/MS following completion of the peptide isolation procedure.The total cell count and the amount of total peptide were calculatedfrom triplicate measurements per tissue sample. The peptide-specificisolation efficiencies were calculated as an average from 10 spikeexperiments each measured as a triplicate (see Example 6 and Table 12).

The present invention provides peptides that are useful in treatingcancers/tumors, preferably RCC that over- or exclusively present thepeptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on primary humanRCC samples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy kidney cells or other normal tissue cells, demonstrating a highdegree of tumor association of the source genes (see Example 2).Moreover, the peptides themselves are strongly over-presented on tumortissue—“tumor tissue” in relation to this invention shall mean a samplefrom a patient suffering from RCC, but not on normal tissues (seeExample 1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. RCC cells presenting the derived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well. Thus, the peptides of the present invention areuseful for generating an immune response in a patient by which tumorcells can be destroyed. An immune response in a patient can be inducedby direct administration of the described peptides or suitable precursorsubstances (e.g. elongated peptides, proteins, or nucleic acids encodingthese peptides) to the patient, ideally in combination with an agentenhancing the immunogenicity (i.e. an adjuvant). The immune responseoriginating from such a therapeutic vaccination can be expected to behighly specific against tumor cells because the target peptides of thepresent invention are not presented on normal tissues in comparable copynumbers, preventing the risk of undesired autoimmune reactions againstnormal cells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are peptides capable of binding to TCRs and antibodies whenpresented by an MHC molecule. The present description also relates tonucleic acids, vectors and host cells for expressing TCRs and peptidesof the present description; and methods of using the same.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to a peptide-HLAmolecule complex with a binding affinity (KD) of about 100 μM or less,about 50 μM or less, about 25 μM or less, or about 10 μM or less. Morepreferred are high affinity TCRs having binding affinities of about 1 μMor less, about 100 nM or less, about 50 nM or less, about 25 nM or less.Non-limiting examples of preferred binding affinity ranges for TCRs ofthe present invention include about 1 nM to about 10 nM; about 10 nM toabout 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM;about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM toabout 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; andabout 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta heterodimeric TCRs of the present description may have a TRACconstant domain sequence and a TRBC1 or TRBC2 constant domain sequence,and the TRAC constant domain sequence and the TRBC1 or TRBC2 constantdomain sequence of the TCR may be linked by the native disulfide bondbetween Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, an peptide-HLA molecule complex, which is atleast double that of a TCR comprising the unmutated TCR alpha chainand/or unmutated TCR beta chain. Affinity-enhancement of tumor-specificTCRs, and its exploitation, relies on the existence of a window foroptimal TCR affinities. The existence of such a window is based onobservations that TCRs specific for HLA-A2-restricted pathogens have KDvalues that are generally about 10-fold lower when compared to TCRsspecific for HLA-A2-restricted tumor-associated self-antigens. It is nowknown, although tumor antigens have the potential to be immunogenic,because tumors arise from the individual's own cells only mutatedproteins or proteins with altered translational processing will be seenas foreign by the immune system. Antigens that are upregulated oroverexpressed (so called self-antigens) will not necessarily induce afunctional immune response against the tumor: T-cells expressing TCRsthat are highly reactive to these antigens will have been negativelyselected within the thymus in a process known as central tolerance,meaning that only T-cells with low-affinity TCRs for self-antigensremain. Therefore, affinity of TCRs or variants of the presentdescription to the peptides according to the invention can be enhancedby methods well known in the art.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with peptide of interest, incubating PBMCs obtained from thetransgenic mice with tetramer-phycoerythrin (PE), and isolating the highavidity T-cells by fluorescence activated cell sorting (FACS)-Caliburanalysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient. In anotheraspect, to obtain T-cells expressing TCRs of the present description,TCR RNAs are synthesized by techniques known in the art, e.g., in vitrotranscription systems. The in vitro-synthesized TCR RNAs are thenintroduced into primary CD8+ T-cells obtained from healthy donors byelectroporation to re-express tumor specific TCR-alpha and/or TCR-betachains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed. In addition to strong promoters, TCRexpression cassettes of the present description may contain additionalelements that can enhance transgene expression, including a centralpolypurine tract (cPPT), which promotes the nuclear translocation oflentiviral constructs (Follenzi et al., 2000), and the woodchuckhepatitis virus posttranscriptional regulatory element (wPRE), whichincreases the level of transgene expression by increasing RNA stability(Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced. (Schmitt et al. 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “op-timal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3ζ fusion). (Schmitt et al. 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 114, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO:1 to SEQ ID NO: 114,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 114, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 114 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:114 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 114, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 114.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of RCC.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 114 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are RCC cells or other solid orhaematological tumor cells such as lung cancer, brain cancer, stomachcancer, colon or rectal cancer, liver cancer, pancreatic cancer,prostate cancer, leukemias, breast cancer, melanoma, ovarian cancer, andesophageal cancer.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of RCC. The present invention also relates to the use of thesenovel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a RCC marker (poly)peptide,delivery of a toxin to a RCC cell expressing a cancer marker gene at anincreased level, and/or inhibiting the activity of a RCC markerpolypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length RCC marker polypeptides or fragments thereof maybe used to generate the antibodies of the invention. A polypeptide to beused for generating an antibody of the invention may be partially orfully purified from a natural source, or may be produced usingrecombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 114polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the RCC marker polypeptide used togenerate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating RCC, the efficacyof the therapeutic antibody can be assessed in various ways well knownto the skilled practitioner. For instance, the size, number, and/ordistribution of cancer in a subject receiving treatment may be monitoredusing standard tumor imaging techniques. A therapeutically-administeredantibody that arrests tumor growth, results in tumor shrinkage, and/orprevents the development of new tumors, compared to the disease coursethat would occurs in the absence of antibody administration, is anefficacious antibody for treatment of cancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it can be expressed in Tcells used for adoptive transfer. Further information can be found in WO2004/033685A1 and WO 2004/074322A1. A combination of sTCRs is describedin WO 2012/056407A1. Additional methods for the production are disclosedin WO 2013/057586A1.

It is a further aspect of the invention to provide a TCR, for example asoluble T-cell receptor (sTCR) recognizing a specific peptide-MHCcomplex as disclosed herein.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S)) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 114 and/or SEQ ID NO:115 to SEQ ID NO: 151 or a variant amino acid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively eliciting high-or low-avidity antigen-specific T cell responses with high efficiencyfrom blood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, andvaccinia-infected target cells. In addition plant viruses may be used(see, for example, Porta et al. (Porta et al., 1994) which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 114.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to normal levels of expression or that thegene is silent in the tissue from which the tumor is derived but in thetumor it is expressed. By “over-expressed” the inventors mean that thepolypeptide is present at a level at least 1.2-fold of that present innormal tissue; preferably at least 2-fold, and more preferably at least5-fold or 10-fold the level present in normal tissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above. Protocols for this so-called adoptive transfer of Tcells are also well known in the art. Reviews can be found in: Gattioniet al. and Morgan et al. (Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and

(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from RCC, themedicament of the invention is preferably used to treat RCC.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue of RCCpatients with various HLA-A HLA-B and HLA-C alleles. It may contain MHCclass I and MHC class II peptides or elongated MHC class I peptides. Inaddition to the tumor associated peptides collected from several RCCtissues, the warehouse may contain HLA-A*02 and HLA-A*24 markerpeptides. These peptides allow comparison of the magnitude of T-cellimmunity induced by TUMAPS in a quantitative manner and hence allowimportant conclusion to be drawn on the capacity of the vaccine toelicit anti-tumor responses. Secondly, they function as importantpositive control peptides derived from a “non-self” antigen in the casethat any vaccine-induced T-cell responses to TUMAPs derived from “self”antigens in a patient are not observed. And thirdly, it may allowconclusions to be drawn, regarding the status of immunocompetence of thepatient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, RCC samples from patients andblood from healthy donors were analyzed in a stepwise approach:

1. HLA ligands from the malignant material were identified by massspectrometry

2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue (RCC)compared with a range of normal organs and tissues

3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromRCC patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients tumor and, wherepossible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (al) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from RCC cells and since it was determined that thesepeptides are not or at lower levels present in normal tissues, thesepeptides can be used to diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for RCC. Presence of groups of peptides can enableclassification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A to 1N show the over-presentation of various peptides in normaltissues and RCC. FIG. 1A) Gene: SLC17A3, Peptide: ALIVSLPYL (SEQ ID NO.:1)—Tissues from left to right: 1 adipose tissues, 3 adrenal glands, 2arteries, 3 bone marrows, 7 brains, 3 breasts, 13 colons, 1 ovary, 1duodenum 4 esophagi, 2 gallbladders, 3 hearts, 4 leukocyte samples, 19livers, 43 lungs, 1 lymph node, 1 ovary, 6 pancreas, 2 peripheralnerves, 1 peritoneum, 1 pituitary gland, 3 pleuras, 1 prostate, 6 recti,3 skeletal muscles, 3 skins, 2 small intestines, 4 spleens, 5 stomachs,1 testis, 2 thymi, 3 thyroid glands, 2 uteri, 2 veins, 12 kidneys, 18RCC. The peptide was also found on liver cancer (not shown). FIG. 1B)Gene: SOGA2, Peptide: YLEEDVYQL (SEQ ID NO.: 128)—Tissues from left toright: 1 adipose tissues, 3 adrenal glands, 2 arteries, 3 bone marrows,7 brains, 3 breasts, 13 colons, 1 ovary, 1 duodenum 4 esophagi, 2gallbladders, 3 hearts, 4 leukocyte samples, 19 livers, 43 lungs, 1lymph node, 1 ovary, 6 pancreas, 2 peripheral nerves, 1 peritoneum, 1pituitary gland, 3 pleuras, 1 prostate, 6 recti, 3 skeletal muscles, 3skins, 2 small intestines, 4 spleens, 5 stomachs, 1 testis, 2 thymi, 3thyroid glands, 2 uteri, 2 veins, 12 kidneys, 18 RCC. The peptide wasalso found on pancreatic cancer, ovarian cancers, stomach cancer, andlung cancer (not shown). FIG. 1C) Gene: SEMASB, Peptide: ALDPSGNQLI (SEQID NO.: 12)—Tissues from left to right: 1 adipose tissues, 3 adrenalglands, 2 arteries, 3 bone marrows, 7 brains, 3 breasts, 13 colons, 1ovary, 1 duodenum 4 esophagi, 2 gallbladders, 3 hearts, 4 leukocytesamples, 19 livers, 43 lungs, 1 lymph node, 1 ovary, 6 pancreas, 2peripheral nerves, 1 peritoneum, 1 pituitary gland, 3 pleuras, 1prostate, 6 recti, 3 skeletal muscles, 3 skins, 2 small intestines, 4spleens, 5 stomachs, 1 testis, 2 thymi, 3 thyroid glands, 2 uteri, 2veins, 12 kidneys, 18 RCC. The peptide was also found on ovarian cancer,brain cancer, and lung cancer (not shown). FIG. 1D) Gene: RGS5, Peptide:GLASFKSFL (SEQ ID NO.: 8)—Tissues from left to right: 1 adipose tissues,3 adrenal glands, 2 arteries, 3 bone marrows, 7 brains, 3 breasts, 13colons, 1 ovary, 1 duodenum 4 esophagi, 2 gallbladders, 3 hearts, 4leukocyte samples, 19 livers, 43 lungs, 1 lymph node, 1 ovary, 6pancreas, 2 peripheral nerves, 1 peritoneum, 1 pituitary gland, 3pleuras, 1 prostate, 6 recti, 3 skeletal muscles, 3 skins, 2 smallintestines, 4 spleens, 5 stomachs, 1 testis, 2 thymi, 3 thyroid glands,2 uteri, 2 veins, 12 kidneys, 18 RCC. The peptide was also found onprostate cancer, breast cancer, colon cancer, liver cancer, melanoma,ovarian cancer, esophageal cancer, pancreatic cancer, brain cancer,stomach cancer and lung cancer (not shown). FIG. 1E) Gene: SLC16A3,Peptide: VVDEGPTGV (SEQ ID NO.: 135)—Tissues from left to right: 1leucocytic cell line, 5 normal tissues (3 lungs, 1 lymph node, 1spleen), 77 cancer tissues (2 brain cancers, 1 breast cancer, 1 coloncancer, 1 esophageal cancer, 10 kidney cancers, 1 leukemia, 1 livercancer, 35 lung cancers, 12 ovarian cancers, 8 pancreatic cancers, 5stomach cancers). Samples without peptide presentation are not shown.The normal (healthy) tissue panel tested was the same as in FIGS. 1A-D.FIG. 1F) Gene:ESM1, Peptide: LLVPAHLVAA (SEQ ID No.: 25)—Tissues fromleft to right: 1 adipose tissue, 3 adrenal glands, 2 arteries, 3 bonemarrows, 7 brains, 3 breasts, 13 colons, 1 duodenum, 4 esophagi, 2gallbladders, 3 hearts, 4 leukocytes, 19 livers, 43 lungs, 1 lymph node,1 ovary, 6 pancreases, 2 peripheral nerves, 1 peritoneum, 1 pituitarygland, 3 pleuras, 1 prostate, 6 rectums, 3 skeletal muscles, 3 skins, 2small intestines, 4 spleens, 5 stomachs, 1 testis, 2 thymi, 3 thyroidglands, 2 uteri, 2 veins, 12 kidneys, 18 RCC. The peptide was also foundon pancreatic cancer, esophageal cancer, brain cancer, lung cancer anduterine cancer (not shown). FIG. 1G) Gene: ARHGAP42, Peptide: ILIKHLVKV(SEQ ID NO.: 15)—Tissues from left to right: 1 cell line (1 melanoma),17 cancer tissues (1 colon cancer, 5 kidney cancers, 1 liver cancer, 3lung cancers, 4 lymph node cancers, 1 testis cancer, 2 uterus cancers).FIG. 1H) Gene: HTR, Peptide: FIADVVEKI (SEQ ID NO.: 33)—Tissues fromleft to right: 16 cancer tissues (1 brain cancer, 1 breast cancer, 1esophageal cancer, 3 kidney cancers, 1 leukocytic leukemia cancer, 5lung cancers, 1 lymph node cancer, 2 ovarian cancers, 1 pancreascancer). FIG. 1I) Gene: HSF2B, Peptide: VLLDTILQL (SEQ ID NO.:38)—Tissues from left to right: 1 benign (kidney cancer), 3 cell-lines(1 pancreas, 1 pleura, 1 prostate), 1 other disease (1 skin), 9 normaltissues (1 lung, 1 lymph node, 2 placentas, 1 small intestine, 3spleens, 1 thyroid gland), 67 cancer tissues (1 bile duct cancer, 5brain cancers, 2 breast cancers, 2 esophageal cancers, 2 gallbladdercancers, 4 kidney cancers, 7 leukocytic leukemia cancers, 3 livercancers, 16 lung cancers, 5 lymph node cancers, 1 myeloid cells cancer,9 ovarian cancers, 1 pancreas cancer, 1 rectum cancer, 5 skin cancers, 2urinary bladder cancers, 1 uterus cancer). FIG. 1J) Gene: TRAM1,Peptide: YLLNLNHLGL (SEQ ID NO.: 39)—Tissues from left to right: 2 celllines (1 kidney, 1 pancreas), 1 normal tissues (1 lung), 19 cancertissues (1 breast cancer, 2 kidney cancers, 3 leukocytic leukemiacancers, 2 liver cancers, 7 lung cancers, 1 lymph node cancer, 1 ovariancancer, 1 rectum cancer, 1 urinary bladder cancer). FIG. 1K) Gene:PXDNL, Peptide: SILDAVQRV (SEQ ID NO.: 52)—Tissues from left to right:23 cancer tissues (3 brain cancers, 4 breast cancers, 3 kidney cancers,8 lung cancers, 2 ovarian cancers, 1 pancreas cancer, 1 skin cancer, 1uterus cancer). FIG. 1L) Gene: THY1, Peptide: SLLQATDFMSL (SEQ ID NO.:82)—Tissues from left to right: 11 cell-lines (11 pancreas cell lines),4 normal tissues (1 kidney, 1 lymph node, 1 placenta, 1 trachea), 36cancer tissues (1 bile duct cancer, 5 brain cancers, 3 breast cancers, 4colon cancers, 1 esophageal cancer, 3 kidney cancers, 1 liver cancer, 9lung cancers, 1 lymph node cancer, 1 ovarian cancer, 2 pancreas cancers,1 rectum cancer, 2 skin cancers, 1 urinary bladder cancer, 1 uteruscancer). FIG. 1M) Gene: ARRDC3, Peptide: KIPPVSPSI (SEQ ID NO.:98)—Tissues from left to right: 2 cell lines (2 kidney), 4 normaltissues (1 adrenal gland, 1 lung, 1 lymph node, 1 placenta), 47 cancertissues (4 brain cancers, 1 breast cancer, 1 esophageal cancer, 1gallbladder cancer, 5 kidney cancers, 1 leukocytic leukemia cancer, 5liver cancers, 12 lung cancers, 2 lymph node cancers, 4 ovarian cancers,3 prostate cancers, 2 skin cancers, 6 uterus cancers). FIG. 1N) Gene:TIMP1, Peptide: KLQDGLLHI (SEQ ID NO.: 103)—Tissues from left to right:1 cell line (1 blood cells), 29 cancer tissues (2 brain cancers, 5 coloncancers, 3 kidney cancers, 1 liver cancer, 4 lung cancers, 2 lymph nodecancers, 3 ovarian cancers, 1 pancreas cancer, 1 rectum cancer, 6 skincancers, 1 testis cancer).

FIGS. 2A to 2D show exemplary expression profiles (relative expressioncompared to normal kidney) of source genes of the present invention thatare highly over-expressed or exclusively expressed in RCC in a panel ofnormal tissues and 35 RCC samples. Tissues from left to right: adrenalgland, artery, bone marrow, brain (whole), breast, colon, esophagus,heart, kidney (triplicate), leukocytes, liver, lung, lymph node, ovary,pancreas, placenta, prostate, salivary gland, skeletal muscle, skin,small intestine, spleen, stomach, testis, thymus, thyroid gland, urinarybladder, uterine cervix, uterus, vein, 23 normal kidney samples, 35 RCCsamples. FIG. 2A) GAL3ST1; FIG. 2B) EGLN3; FIG. 2C) APOL1; and FIG. 2D)MET.

FIGS. 3A-3F show exemplary immunogenicity data: flow cytometry resultsafter peptide-specific multimer staining, as results of peptide-specificin vitro CD8+ T cell responses of a healthy HLA-A*02+ donor. CD8+ Tcells were primed using artificial APCs coated with anti-CD28 mAb andHLA-A*02 in complex with SeqID No 127 peptide (FIG. 3A, left panel),SeqID No 145 peptide (FIG. 3B, left panel), SeqID No 20 peptide (FIG.3C, left panel), SeqID No 34 peptide (FIG. 3D, left panel), SeqID No 1peptide (FIG. 3E, left panel) or SeqID No 15 peptide (FIG. 3F, leftpanel) respectively. After three cycles of stimulation, the detection ofpeptide-reactive cells was performed by 2D multimer staining withA*02/FZD-001 (SeqID No 127) (FIG. 3A), A*02/ATP-001 (SeqID No 145) (FIG.3B), A*02/SeqID No 20 (FIG. 3C), A*02/SeqID No 34 (FIG. 3D), A*02/SeqIDNo 1 (FIG. 3E) or A*02/SeqID No 15 (FIG. 3F). Right panels (FIGS. 3C,3D, 3E and 3F) show control staining of cells stimulated with irrelevantA*02/peptide complexes. Viable singlet cells were gated for CD8+lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+ cells among CD8+ lymphocytes are indicated.

EXAMPLES Example 1

Identification and Quantitation of Tumor Associated Peptides Presentedon the Cell Surface

Tissue Samples

Patients' tumor tissues were obtained from BioServe (Beltsville, Md.,USA); University Hospital of Munich; Kyoto Prefectural University ofMedicine (KPUM); University Hospital of Tübingen.

Normal (healthy) tissues were obtained from Bio-Options Inc., CA, USA;BioServe, Beltsville, Md., USA; Capital BioScience Inc., Rockville, Md.,USA; Geneticist Inc., Glendale, Calif., USA; University Hospital ofGeneva; University Hospital of Heidelberg; Kyoto Prefectural Universityof Medicine (KPUM); University Hospital Munich; ProteoGenex Inc., CulverCity, Calif., USA; University Hospital of Tübingen.

Written informed consents of all patients had been given before surgeryor autopsy. Tissues were shock-frozen immediately after excision andstored until isolation of TUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, —B, C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOP5 strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the Orbitrap(R=30 000), which was followed by MS/MS scans also in the Orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus each identified peptidecan be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofiles juxtapose RCC samples to a baseline of normal tissue samples.Presentation profiles of exemplary over-presented peptides are shown inFIGS. 1A-1N. Presentation scores for exemplary peptides are shown inTable 8.

TABLE 8 Presentation scores. The table lists peptides thatare very highly over-presented on tumors comparedto a panel of normal tissues (+++), highlyover-presented on tumors compared to a panel ofnormal tissues (++) or over-presented ontumors compared to a panel of normal tissues (+). SEQ ID Peptide No.Sequence Presentation 1 ALIVSLPYL +++ 2 ILWREVVTL +++ 3 RLLGEVQAL +++ 4FLSQDIITV +++ 5 YLYPNLTRL +++ 6 VLFELSKTV +++ 7 FLLSLIDRL +++ 8GLASFKSFL +++ 9 ILLQKPDSV +++ 10 KLLQNNYGL + 11 FIQTEAPKEV +++ 12ALDPSGNQLI +++ 13 KIMAQILTV + 14 ALLTETIFL ++ 15 ILIKHLVKV +++ 16FMPEELPQL +++ 17 ILAQQVHAL +++ 18 YVLDLAAKV +++ 19 LLDPGSLQL + 20AVANTTFTV +++ 21 RLIQGDQILSV +++ 23 YIQEVVQYI +++ 24 FTLGTTVFL ++ 26SLMEILYTL +++ 27 SLSDLLVSL ++ 28 FIADLVVGL ++ 29 ILLDLEQAL + 30QLFYTKIFL ++ 31 VLFGLDPAVIKV + 32 FLAGGIRGSGA +++ 33 FIADVVEKI +++ 34ELNNQNFYL +++ 35 VLHSLQTQL +++ 36 SLFGKKYIL +++ 37 VLAPVILML +++ 38VLLDTILQL +++ 39 YLLNLNHLGL +++ 40 YIQEHLLQI ++ 41 GLLKTLQKL + 42VILDTGTIQL +++ 43 YLKDELDEL +++ 44 ALFSFVTAL + 45 ALLGIPLTL ++ 47TLAEVRAVQEI + 48 VVASNIMEV +++ 49 VLIVEVPGV +++ 50 SLSDHIVLL + 51NLWPMILTL +++ 52 SILDAVQRV +++ 55 YLALILPVL +++ 56 ILMDFSNSM + 57SLQKEILYL +++ 58 FLVDFEQSHL ++ 60 ILWKDIEYV +++ 61 SLMGILLRI +++ 62VLAGPAFLVQL +++ 66 TLLKTIIKV ++ 67 LLDVLAPLV + 68 YVLTQPPSV +++ 69ILADLLPSL ++ 70 SLTALRLLL + 72 YSLEKVFGI + 73 GLDGIPFTV ++ 74GLFHKQVTV + 75 FLIKSINLV ++ 77 SLIKHKIML ++ 78 ALLDTVVQA + 79ALADIVWRA + 80 KLASMLETL + 83 IQWSIVPEV ++ 86 GLLQGKLALL + 88TLAELHISL + 89 SLLLAVTEV + 90 FTLEKNFVI ++ 91 MLLSSLVSL + 92 FLFRDILEL+++ 93 GVMAGDIYSV ++ 94 ILHHKVYDL + 96 TLAETLVNL + 97 TLISELVQA + 98KIPPVSPSI +++ 99 GLAPHLEQI + 100 KLNVAPLAV + 101 HIYDKAFITV ++ 102LLFDVHTTL + 103 KLQDGLLHI +++ 104 ALFEGVVRQI +++ 105 ALADLDELLIRA ++ 106VLMDLKALL + 107 VLMDLKALLL ++ 108 VLISVLQAI ++ 109 YLWSRVEKL + 110LLDLHSYLL + 111 TLLETEMLL +++ 112 LLFDHLEPIEL + 113 SLFDWNVKL ++ 114ALAVNISAA ++ 115 LLDPKTIFL +++ 116 GLVDIMVHL +++ 117 VLFGELPAL +++ 118FLNAIETAL +++ 119 RLHDENILL +++ 120 GLAGDNIYL +++ 121 ALLRTVVSV ++ 122SLDPSSPQV ++ 123 YVDPVITSI ++ 124 ILSPLSVAL + 125 KLDPTKTTL +++ 126KIQEILTQV +++ 127 VLAPLFVYL ++ 128 YLEEDVYQL +++ 129 VLAPRVLRA ++ 130ALPTVLVGV ++ 131 VMAGDIYSV ++ 132 SVASTITGV ++ 133 QLIDYERQL ++ 134VADKIHSV +++ 135 VVDEGPTGV +++ 136 YQDPHSTAV ++ 137 TLVAIVVGV ++ 138SLDTLMTYV ++ 139 ILNVDGLIGV ++ 140 SLANNVTSV ++ 141 LLVDDSFLHTV +++ 143ALFVRLLALA ++ 145 SLHFLILYV + 150 TLMPNINKL +++ 151 YMYEGPAPRI +++

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues was obtained commercially (Ambion,Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam,Netherlands; BioChain, Hayward, Calif., USA). The RNA from severalindividuals (between 2 and 123 individuals) was mixed such that RNA fromeach individual was equally weighted.

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

Microarray Experiments

Gene expression analysis of all tumor and normal tissue RNA samples wasperformed by Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0oligonucleotide microarrays (Affymetrix, Santa Clara, Calif., USA). Allsteps were carried out according to the Affymetrix manual. Briefly,double-stranded cDNA was synthesized from 5-8 μg of total RNA, usingSuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biotech,Ebersberg, Germany) as described in the manual. In vitro transcriptionwas performed with the BioArray High Yield RNA Transcript Labelling Kit(ENZO Diagnostics, Inc., Farmingdale, N.Y., USA) for the U133A arrays orwith the GeneChip IVT Labelling Kit (Affymetrix) for the U133 Plus 2.0arrays, followed by cRNA fragmentation, hybridization, and staining withstreptavidin-phycoerythrin and biotinylated anti-streptavidin antibody(Molecular Probes, Leiden, Netherlands). Images were scanned with theAgilent 2500A GeneArray Scanner (U133A) or the Affymetrix Gene-ChipScanner 3000 (U133 Plus 2.0), and data were analyzed with the GCOSsoftware (Affymetrix), using default settings for all parameters. Fornormalization, 100 housekeeping genes provided by Affymetrix were used.Relative expression values were calculated from the signal log ratiosgiven by the software and the normal kidney sample was arbitrarily setto 1.0. Exemplary expression profiles of source genes of the presentinvention that are highly over-expressed or exclusively expressed in RCCare shown in FIGS. 2A-2D. Expression scores for further exemplary genesare shown in Table 9.

TABLE 9 Expression scores. The table lists peptides fromgenes that are very highly over-expressed intumors compared to a panel of normal tissues(+++), highly over-expressed in tumorscompared to a panel of normal tissues (++) orover-expressed in tumors compared to a panel of normal tissues (+). SeqGene ID Expres- NO Gene Name sequence sion 1 SLC17A3 ALIVSLPYL ++ 2 HSF4ILWREVVTL ++ 3 HSF4 RLLGEVQAL ++ 6 ATP11A VLFELSKTV + 7 EGLN3 FLLSLIDRL++ 18 ACLY YVLDLAAKV + 28 ENO1, ENO2, ENO3 FIADLVVGL + 35 ANGPTL4VLHSLQTQL ++ 37 ABCC3 VLAPVILML + 40 IVNS1ABP YIQEHLLQI + 50 ITGA3SLSDHIVLL + 51 ITGA3 NLWPMILTL + 55 SLC16A4 YLALILPVL + 59 TGFBISLKNNVVSV + 62 SLC47A1 VLAGPAFLVQL + 63 ERAP1 GLIEDHFDVTV + 65TPI1, TPI1P1 IIYGGSVTGA + 76 PDZK1P2, PDZK1, VLADDHLIEV ++ PDZK1P1 81LRP2 SLLPALPKL + 87 APOL1 LADGVQKV ++ 93 PLIN2 GVMAGDIYSV + 94 CYB5AILHHKVYDL + 101 FLT1 HIYDKAFITV ++ 105 HSPG2 ALADLDELLIRA + 115 HAVCR1LLDPKTIFL + 120 SLC22A2 GLAGDNIYL + 122 GAL3ST1 SLDPSSPQV +++ 123 METYVDPVITSI ++ 125 NDRG1 KLDPTKTTL + 127 FZD2, FZD1, FZD7 VLAPLFVYL + 131PLIN2 VMAGDIYSV + 132 PLIN2 SVASTITGV + 137 FKBP10 TLVAIVVGV + 139 ACLYILNVDGLIGV + 143 TGFBI ALFVRLLALA +

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for 22 HLA-A*0201 restricted TUMAPsof the invention so far, demonstrating that these peptides are T-cellepitopes against which CD8+ precursor T cells exist in humans (Table10).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nürnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 152) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5 (SEQ ID NO.153)), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oreg., USA). In vitro priming of specificmultimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for RCC Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for 2peptides of the invention are shown in FIGS. 3A-3F together withcorresponding negative controls. Results for 3 peptides from theinvention are summarized in Table 10A) and B).

TABLE 10A in vitro immunogenicity of HLA class I peptides ofthe invention Exemplary results of in vitro immunogenicityexperiments conducted by the applicant for thepeptides of the invention. <20% = +; 20%-49% = ++; 50%-69% = +++; ≥70% =++++ Seq ID Peptide ID wells donors 123 YVDPVITSI + ++++ 127 VLAPLFVYL++ ++++ 143 ALFVRLLALA + +++

TABLE 10B In vitro immunogenicity of HLA class I peptides ofthe invention Exemplary results of in vitro immunogenicityexperiments conducted by the applicant forpeptides of the invention. Results of in vitroimmunogenicity experiments are indicated.Percentage of positive wells and donors (amongevaluable) are summarized as indicated <20% = +; 20%-49% = ++; 50%-69% =+++; ≥70% = ++++ SEQ ID No Sequence Wells positive [%] 1 ALIVSLPYL + 2ILWREVVTL + 3 RLLGEVQAL + 6 VLFELSKTV + 7 FLLSLIDRL + 9 ILLQKPDSV + 15ILIKHLVKV + 16 FMPEELPQL + 18 YVLDLAAKV ++ 20 AVANTTFTV + 22 FLSPPLPSV++ 29 ILLDLEQAL + 34 ELNNQNFYL ++ 36 SLFGKKYIL ++ 94 ILHHKVYDL ++ 95KLTDVGIATL ++++ 98 KIPPVSPSI ++++

Example 4

Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizates (trifluoro acetate salt) in purities of >50%. All TUMAPsare preferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5

MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1 hat 37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100 fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1 h at 37° C., washed again and detected with TMB solutionthat is stopped with NH₂SO₄. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 11 MHC class I binding scores.Binding of HLA-class I restricted peptides to HLA-A*02:01 was ranged by peptide exchange yield: ≥10% = +; ≥20% = ++; ≥50 =+++; ≥75% = ++++ Seq ID Sequence Peptide exchange 1 ALIVSLPYL +++ 2ILWREVVTL +++ 3 RLLGEVQAL ++ 4 FLSQDIITV +++ 5 YLYPNLTRL ++ 6 VLFELSKTV+++ 7 FLLSLIDRL +++ 8 GLASFKSFL ++ 9 ILLQKPDSV ++ 10 KLLQNNYGL ++ 11FIQTEAPKEV ++ 12 ALDPSGNQLI ++ 13 KIMAQILTV +++ 14 ALLTETIFL ++ 15ILIKHLVKV ++ 16 FMPEELPQL ++ 17 ILAQQVHAL +++ 18 YVLDLAAKV +++ 19LLDPGSLQL ++ 20 AVANTTFTV ++ 21 RLIQGDQILSV ++ 22 FLSPPLPSV +++ 23YIQEVVQYI +++ 24 FTLGTTVFL +++ 25 LLVPAHLVAA ++ 27 SLSDLLVSL +++ 28FIADLVVGL ++ 29 ILLDLEQAL +++ 30 QLFYTKIFL ++ 31 VLFGLDPAVIKV +++ 33FIADVVEKI ++ 34 ELNNQNFYL +++ 35 VLHSLQTQL ++ 36 SLFGKKYIL ++ 37VLAPVILML ++ 38 VLLDTILQL +++ 39 YLLNLNHLGL +++ 40 YIQEHLLQI +++ 41GLLKTLQKL ++ 42 VILDTGTIQL ++ 43 YLKDELDEL +++ 44 ALFSFVTAL +++ 45ALLGIPLTL +++ 46 GLSEVLVQI +++ 47 TLAEVRAVQEI +++ 48 VVASNIMEV ++ 49VLIVEVPGV +++ 50 SLSDHIVLL ++ 51 NLWPMILTL +++ 52 SILDAVQRV ++ 53FLLEIRQTL ++ 54 ALVAKGLVQA ++ 55 YLALILPVL ++ 56 ILMDFSNSM +++ 57SLQKEILYL ++ 58 FLVDFEQSHL ++++ 59 SLKNNVVSV +++ 60 ILWKDIEYV +++ 61SLMGILLRI ++ 62 VLAGPAFLVQL +++ 63 GLIEDHFDVTV ++ 64 LLAASVALA ++ 65IIYGGSVTGA ++ 66 TLLKTIIKV ++ 67 LLDVLAPLV ++ 68 YVLTQPPSV ++ 69ILADLLPSL ++ 70 SLTALRLLL ++ 71 ALDGHLYAV +++ 72 YSLEKVFGI ++ 73GLDGIPFTV +++ 74 GLFHKQVTV +++ 75 FLIKSINLV +++ 76 VLADDHLIEV ++ 77SLIKHKIML ++ 78 ALLDTVVQA ++ 79 ALADIVWRA +++ 80 KLASMLETL +++ 81SLLPALPKL ++ 82 SLLQATDFMSL +++ 83 IQWSIVPEV +++ 84 YLMDEGAHL ++ 85FVMSEIRTV +++ 86 GLLQGKLALL ++ 88 TLAELHISL ++ 89 SLLLAVTEV ++ 90FTLEKNFVI ++ 91 MLLSSLVSL ++ 92 FLFRDILEL +++ 93 GVMAGDIYSV ++++ 94ILHHKVYDL ++ 95 KLTDVGIATL ++ 96 TLAETLVNL +++ 97 TLISELVQA ++ 98KIPPVSPSI ++ 99 GLAPHLEQI ++ 100 KLNVAPLAV ++ 101 HIYDKAFITV ++ 102LLFDVHTTL +++ 103 KLQDGLLHI ++ 104 ALFEGVVRQI +++ 105 ALADLDELLIRA +++106 VLMDLKALL +++ 107 VLMDLKALLL +++ 108 VLISVLQAI ++ 109 YLWSRVEKL ++++110 LLDLHSYLL ++ 111 TLLETEMLL ++ 112 LLFDHLEPIEL +++ 113 SLFDWNVKL +++114 ALAVNISAA +++

Example 6

Absolute Quantitation of Tumor Associated Peptides Presented on the CellSurface

The generation of binders, such as antibodies and/or TCRs, is alaborious process, which may be conducted only for a number of selectedtargets. In the case of tumor-associated and—specific peptides,selection criteria include but are not restricted to exclusiveness ofpresentation and the density of peptide presented on the cell surface.The quantitation of TUMAP copies per cell in solid tumor samplesrequires the absolute quantitation of the isolated TUMAP, the efficiencyof TUMAP isolation, and the cell count of the tissue sample analyzed.

Peptide Quantitation by nanoLC-MS/MS

For an accurate quantitation of peptides by mass spectrometry, acalibration curve was generated for each peptide using the internalstandard method. The internal standard is a double-isotope-labelledvariant of each peptide, i.e. two isotope-labelled amino acids wereincluded in TUMAP synthesis. It differs from the tumor-associatedpeptide only in its mass but shows no difference in otherphysicochemical properties (Anderson et al., 2012). The internalstandard was spiked to each MS sample and all MS signals were normalizedto the MS signal of the internal standard to level out potentialtechnical variances between MS experiments.

The calibration curves were prepared in at least three differentmatrices, i.e. HLA peptide eluates from natural samples similar to theroutine MS samples, and each preparation was measured in duplicate MSruns. For evaluation, MS signals were normalized to the signal of theinternal standard and a calibration curve was calculated by logisticregression.

For the quantitation of tumor-associated peptides from tissue samples,the respective samples were also spiked with the internal standard; theMS signals were normalized to the internal standard and quantified usingthe peptide calibration curve.

Efficiency of Peptide/MHC Isolation

As for any protein purification process, the isolation of proteins fromtissue samples is associated with a certain loss of the protein ofinterest. To determine the efficiency of TUMAP isolation, peptide/MHCcomplexes were generated for all TUMAPs selected for absolutequantitation. To be able to discriminate the spiked from the naturalpeptide/MHC complexes, single-isotope-labelled versions of the TUMAPswere used, i.e. one isotope-labelled amino acid was included in TUMAPsynthesis. These complexes were spiked into the freshly prepared tissuelysates, i.e. at the earliest possible point of the TUMAP isolationprocedure, and then captured like the natural peptide/MHC complexes inthe following affinity purification. Measuring the recovery of thesingle-labelled TUMAPs therefore allows conclusions regarding theefficiency of isolation of individual natural TUMAPs.

The efficiency of isolation was analyzed in a low number of samples andwas comparable among these tissue samples. In contrast, the isolationefficiency differs between individual peptides. This suggests that theisolation efficiency, although determined in only a limited number oftissue samples, may be extrapolated to any other tissue preparation.However, it is necessary to analyze each TUMAP individually as theisolation efficiency may not be extrapolated from one peptide to others.

Determination of the Cell Count in Solid, Frozen Tissue

In order to determine the cell count of the tissue samples subjected toabsolute peptide quantitation, the inventors applied DNA contentanalysis. This method is applicable to a wide range of samples ofdifferent origin and, most importantly, frozen samples (Alcoser et al.,2011; Forsey and Chaudhuri, 2009; Silva et al., 2013). During thepeptide isolation protocol, a tissue sample is processed to a homogenouslysate, from which a small lysate aliquot is taken. The aliquot isdivided in three parts, from which DNA is isolated (QiaAmp DNA Mini Kit,Qiagen, Hilden, Germany). The total DNA content from each DNA isolationis quantified using a fluorescence-based DNA quantitation assay (QubitdsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany) in at leasttwo replicates.

In order to calculate the cell number, a DNA standard curve fromaliquots of single healthy blood cells, with a range of defined cellnumbers, has been generated. The standard curve is used to calculate thetotal cell content from the total DNA content from each DNA isolation.The mean total cell count of the tissue sample used for peptideisolation is extrapolated considering the known volume of the lysatealiquots and the total lysate volume.

Peptide Copies Per Cell

With data of the aforementioned experiments, the inventors calculatedthe number of TUMAP copies per cell by dividing the total peptide amountby the total cell count of the sample, followed by division throughisolation efficiency. Copy cell numbers for selected peptides are shownin Table 12.

TABLE 12 Absolute copy numbers. The table lists the results of absolutepeptide quantitation in NSCLC tumor samples. The median number of copiesper cell are indicated for each peptide: <100 = +; >=100 = ++; >=1,000+++; >=10,000 = ++++. The number of samples, in which evaluable, highquality MS data are available, is indicated. SEQ ID Copies per cellNumber of No. Peptide Code (median) samples 1 SLC17A3-001 ++ 18

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The invention claimed is:
 1. A method of eliciting an immune response ina patient who has cancer, comprising administering to said patient apopulation of activated T cells that selectively recognize cancer cellsthat aberrantly present a peptide consisting of the amino acid sequenceof TLMPNINKL (SEQ ID NO: 150), wherein said cancer is selected from thegroup consisting of brain cancer, stomach cancer, liver cancer,leukemias, melanoma, and esophageal cancer.
 2. The method of claim 1,wherein the T cells are autologous to the patient.
 3. The method ofclaim 1, wherein the T cells are obtained from a healthy donor.
 4. Themethod of claim 1, wherein the T cells are obtained from tumorinfiltrating lymphocytes or peripheral blood mononuclear cells.
 5. Themethod of claim 1, wherein the activated T cells are expanded in vitro.6. The method of claim 1, wherein the population of activated T cellsare administered in the form of a composition.
 7. The method of claim 6,wherein the composition further comprises an adjuvant.
 8. The method ofclaim 7, wherein the adjuvant is selected from the group consisting ofanti-CD40 antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide,sunitinib, bevacizumab, interferon-alpha, interferon-beta, CpGoligonucleotides and derivatives, poly-(I:C) and derivatives, RNA,sildenafil, particulate formulations with poly(lactide coglycolide)(PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13,IL-15, IL-21, and IL-23.
 9. The method of claim 1, wherein the activatedT cells are cytotoxic T cells produced by contacting T cells with anantigen presenting cell that presents the peptide in a complex with anMEW class I molecule on the surface of the antigen presenting cell, fora period of time sufficient to activate said T cell.
 10. The method ofclaim 9, wherein the contacting is in vitro.
 11. The method of claim 1,wherein the cancer is liver cancer.
 12. The method of claim 1, whereinthe cancer is melanoma.
 13. A method of eliciting an immune response ina patient who has esophageal cancer, comprising administering to saidpatient a population of activated T cells that selectively recognizecancer cells that aberrantly present a peptide consisting of the aminoacid sequence of TLMPNINKL (SEQ ID NO: 150).
 14. The method of claim 1,wherein the cancer is brain cancer.
 15. The method of claim 1, whereinthe activated T cells release a cytokine.
 16. The method of claim 1,wherein the immune response is capable of killing cancer cells thatpresent a peptide consisting of the amino acid sequence of TLMPNINKL(SEQ ID NO: 150).
 17. A method of treating a patient who has cancer,comprising administering to said patient a population of activated Tcells that selectively recognize cancer cells that aberrantly present apeptide consisting of the amino acid sequence of TLMPNINKL (SEQ ID NO:150); wherein said cancer is selected from the group consisting of braincancer, stomach cancer, liver cancer, leukemias, melanoma, andesophageal cancer.
 18. The method of claim 17, wherein the T cellresponse is a cytotoxic T cell response.
 19. The method of claim 17,wherein the activated T cells are cytotoxic T cells produced bycontacting T cells with an antigen presenting cell that presents thepeptide in a complex with an WIC class I molecule on the surface of theantigen presenting cell, for a period of time sufficient to activatesaid T cell.
 20. The method of claim 19, wherein the contacting is invitro.